JP2012516339A - Exogenous stimulated controlled release material body and use thereof - Google Patents

Abstract

In accordance with the present disclosure, a cross-linked substance comprising a conjugate comprising a multivalent cross-linking agent that binds to an exogenous target molecule, and two or more individual affinity ligands bound to a conjugate framework. These affinity ligands compete with the exogenous target molecule for binding to the cross-linking agent, and as a result of non-covalent interactions between the affinity ligand on different conjugates and the cross-linking agent within the substance. A cross-linked material is provided in which the conjugate is cross-linked. The conjugate also includes a drug.

Description

RELATED APPLICATIONS This application includes US Provisional Application No. 61 / 147,878, filed Jan. 28, 2009, US Provisional Application No. 61 / 159,643, filed Mar. 12, 2009, US Provisional Application No. 61 / 162,107, filed March 20, 2009, US Provisional Application No. 61 / 162,053, filed March 20, 2009, March 20, 2009 US Provisional Application No. 61 / 162,058, US Provisional Application No. 61 / 162,084, filed Mar. 20, 2009, US Patent, filed Mar. 20, 2009 Provisional Application No. 61 / 162,092, U.S. Provisional Application No. 61 / 162,105 filed on March 20, 2009, U.S. Provisional Application No. 61/163 filed on March 25, 2009 , 084, US provisional application 61 / 219,897 filed on June 24, 2009, US provisional application 61 / 223,572, filed July 7, 2009, and October 19, 2009 And claims priority to US Provisional Patent Application No. 61 / 252,857, both of which are hereby incorporated by reference in their entirety.

  Most “controlled release” drug delivery systems work by delaying or delaying the release of the drug after administration. Such systems are useful for some specific types of drugs (eg, due to fewer peaks and grooves in the serum profile, reduced side effects, etc.), but more complex release profiles (eg, , Release proportional to endogenous substances such as glucose, pulsatile release at fixed or variable time points, etc.) is not suitable. For example, treatment of diabetes mellitus with injectable insulin is a well-known test case where slow and slow release of insulin is not effective. Indeed, it is clear that simple supplementation of hormones is not sufficient to prevent the morbidity associated with this disease. The occurrence of such sequelae seems to reflect the failure to provide exogenous insulin (ie, a true “controlled release” system) in line with blood glucose concentration fluctuations within the patient's body. It is done. As a result, there is a need in the art for alternative controlled release drug delivery systems, particularly systems that can be controlled after administration. The present disclosure provides such a system.

  In one aspect, in accordance with the present disclosure, a cross-linked material comprising a conjugate comprising a multivalent cross-linking agent that binds to an exogenous target molecule and two or more individual affinity ligands bound to a conjugate framework. The two or more affinity ligands compete with the exogenous target molecule for binding to the cross-linking agent, and as a result of non-covalent interactions between the affinity ligand on different conjugates and the cross-linking agent, Provided is a crosslinked material in which the conjugate is crosslinked in the material. The conjugate also includes a drug. The drug and affinity ligand may be covalently or non-covalently bound to the conjugate framework. In general, such substances are designed such that the release of the conjugate is triggered by an increase in the local concentration of the exogenous target molecule. The present disclosure also provides a method of using the substance, wherein a stimulating amount of an exogenous target molecule is administered to a patient who has previously been administered the substance of the present disclosure. In addition, the present disclosure provides a method for producing the substance body. In another aspect, the present disclosure provides exemplary cross-linked substances and exogenous target molecules.

Definitions Definitions of specific functional groups, chemical terms, and general terms used throughout this specification are described in more detail below. For the interpretation of the present invention, chemical elements are specified according to the periodic table of elements (CAS version) described in the inner cover of Handbook of Chemistry and Physics, 75th edition, and specific functional groups are generally defined in the above documents. Has been. In addition, the general principles of organic chemistry and specific functional moieties and reactivities are described in Organic Chemistry, Thomas Sorell, University Science Books, Sausalito, 1999; Smith and March Manchester '5th edition. & Sons, Inc. New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc. New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987. Carryers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987.

Acyl—As used herein, the term “acyl” has the general formula —C (═O) R ^X1 , —C (═O) OR ^X1 , —C (═O) —O—C (═O) R. ^X1 , -C (= O) ^SRX1 , -C (= O) N ( ^RX1 ) ₂ , -C (= S) ^RX1 , -C (= S) N ( ^RX1 ) ₂ , and -C ( = S) S (R ^X1 ), -C (= NR ^X1 ) R ^X1 , -C (= NR ^X1 ) OR ^X1 , -C (= NR ^X1 ) SR ^X1 , and -C (= NR ^X1 ) N (R ^{X 1} ) ₂ (wherein R ^X1 is hydrogen; halogen; substituted or unsubstituted hydroxyl; substituted or unsubstituted thiol; substituted or unsubstituted amino; substituted or unsubstituted acyl; cyclic or acyclic, substituted Or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted Branched or unbranched heteroaliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkyl; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkenyl; substituted or non-branched Substituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, aliphatic oxy, heteroaliphatic oxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphatic thoxy, heteroaliphatic thoxy Alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, mono- or dialiphatic amino, mono- or diheteroaliphatic amino, mono- or dialkylamino, mono- or diheteroalkylamino, mono- or Jary Amino, or mono - or di-heteroarylamino a is either; refers to or two R ^X1 groups groups having form a heterocyclic ring of 5-6 membered together). Exemplary acyl groups include aldehyde (—CHO), carboxylic acid (—CO ₂ H), ketone, acyl halide, ester, amide, imine, carbonate, carbamate, and urea. Acyl substituents include, but are not limited to, stable moieties (eg, aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, Amino, azide, nitro, hydroxyl, thiol, halo, aliphatic amino, heteroaliphatic amino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphatic oxy, heteroaliphatic oxy, Alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphatic thioxy, heteroaliphatic thioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, etc. It may be further substituted, include any substituents described herein that result in the formation of Otherwise it is not necessary).

  Aliphatic—As used herein, the term “aliphatic” or “aliphatic group” can be straight-chain (ie, unbranched), branched or cyclic (“carbocyclic”) and is fully saturated. Represents an optionally substituted hydrocarbon moiety that may be present or contain one or more unsaturated units, but is not aromatic. Unless otherwise specified, aliphatic groups contain 1-12 carbon atoms. In some embodiments, aliphatic groups contain 1-6 carbon atoms. In some embodiments, aliphatic groups contain 1-4 carbon atoms, and in other embodiments, aliphatic groups contain 1-3 carbon atoms. Suitable aliphatic groups include, but are not limited to, linear or branched alkyl, alkenyl and alkynyl groups, and hybrids thereof (such as (cycloalkyl) alkyl, (cycloalkenyl) alkyl or (cycloalkyl) alkenyl). .

  Alkenyl—As used herein, the term “alkenyl” is any derivative derived from the removal of one hydrogen atom from a straight or branched aliphatic moiety having at least one carbon-carbon double bond. Represents a monovalent group optionally substituted. In some specific embodiments, the alkenyl group employed in the invention contains 2-6 carbon atoms. In some specific embodiments, the alkenyl group employed in the invention contains 2-5 carbon atoms. In some embodiments, the alkenyl group employed in the invention contains 2-4 carbon atoms. In another embodiment, the alkenyl group employed contains 2-3 carbon atoms. Examples of the alkenyl group include ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl and the like.

  Alkyl—As used herein, the term “alkyl” refers to an optionally substituted straight chain derived from the removal of one hydrogen atom from an aliphatic moiety containing from 1 to 6 carbon atoms, or A branched chain saturated hydrocarbon group. In some embodiments, the alkyl group employed in the invention contains 1-5 carbon atoms. In another embodiment, the alkyl group employed contains 1-4 carbon atoms. In still other embodiments, the alkyl group contains 1-3 carbon atoms. In yet another embodiment, the alkyl group contains 1-2 carbons. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, sec-pentyl, iso-pentyl, tert-butyl, n-pentyl, Neopentyl, n-hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl and the like can be mentioned.

  Alkynyl—As used herein, the term “alkynyl” is optionally derived by the removal of one hydrogen atom from a straight or branched aliphatic moiety having at least one carbon-carbon triple bond. A monovalent group which is substituted. In some specific embodiments, the alkynyl group employed in the invention contains 2-6 carbon atoms. In some specific embodiments, the alkynyl group employed in the invention contains 2-5 carbon atoms. In some embodiments, the alkynyl group employed in the invention contains 2-4 carbon atoms. In another embodiment, the alkynyl group employed contains 2-3 carbon atoms. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

  Aryl—As used herein, the term “aryl” used alone or as part of a larger moiety such as “aralkyl”, “aralkoxy” or “aryloxyalkyl” refers to a total of 5-10 rings Optionally substituted monocyclic and bicyclic, wherein at least one ring in the system is aromatic and each ring in the system contains 3 to 7 ring members The ring system. The term “aryl” may be used interchangeably with the term “aryl ring”. In some specific embodiments of the invention, “aryl” includes, but is not limited to, phenyl, biphenyl, naphthyl, anthracyl, etc., an aromatic ring system that may have one or more substituents. Say.

  Arylalkyl—As used herein, the term “arylalkyl” refers to an alkyl group substituted with an aryl group (eg, an aromatic or heteroaromatic group).

Divalent hydrocarbon chain—As used herein, the term “divalent hydrocarbon chain” (also referred to as “divalent alkylene group”) is a polymethylene group, ie, — (CH ₂ ) _Z —, In the formula, z is 1-30, 1-20, 1-12, 1-8, 1-6, 1-4, 1-3, 1-2, 2-30, 2-20, 2-10, It is a positive integer of 2-8, 2-6, 2-4, or 2-3. A substituted divalent hydrocarbon chain is a polymethylene group in which one or more methylene hydrogen atoms have been replaced with a substituent. Suitable substituents include those described below for substituted aliphatic groups.

  Carbonyl—As used herein, the term “carbonyl” refers to a monovalent or divalent moiety containing a carbon-oxygen double bond. Non-limiting examples of carbonyl groups include aldehydes, ketones, carboxylic acids, esters, amides, enones, acyl halides, anhydrides, ureas, carbamates, carbonates, thioesters, lactones, lactams, hydroxamates, isocyanates, and chloro Formate.

  Alicyclic—As used herein, the term “alicyclic”, “carbocyclic”, or “carbocyclic” is used alone or as part of a larger moiety and is a 3-10 membered book. Refers to an optionally substituted saturated or partially unsaturated, cyclic, aliphatic, monocyclic or bicyclic ring system as described in the specification. Alicyclic groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, and cyclooctadienyl. In some embodiments, the cycloalkyl has 3-6 carbons.

  Halogen—As used herein, the terms “halo” and “halogen” refer to fluorine (fluoro, —F), chlorine (chloro, —Cl), bromine (bromo, —Br), and iodine (iodo, —I). ).

  Heteroaliphatic—As used herein, the term “heteroaliphatic” or “heteroaliphatic group” has 1 to 5 heteroatoms in addition to the carbon atom, and is linear (ie, unbranched) May be branched or cyclic ("heterocyclic"), may be fully saturated, may contain one or more unsaturated units, but may be aromatic Represents an optionally substituted hydrocarbon moiety. Unless otherwise specified, heteroaliphatic groups contain from 1 to 6 carbon atoms, and 1 to 3 carbon atoms are optionally replaced independently with heteroatoms selected from oxygen, nitrogen and sulfur. It has been. In some embodiments, the heteroaliphatic group contains 1-4 carbon atoms, and 1-2 carbon atoms are optionally independently a heteroatom selected from oxygen, nitrogen, and sulfur. Has been replaced. In still other embodiments, the heteroaliphatic group contains 1-3 carbon atoms, and one carbon atom is optionally replaced independently with a heteroatom selected from oxygen, nitrogen, and sulfur. ing. Suitable heteroaliphatic groups include, but are not limited to, linear or branched heteroalkyl, heteroalkenyl, and heteroalkynyl groups.

  Heteroaralkyl—As used herein, the term “heteroaralkyl” refers to an alkyl group substituted with a heteroaryl, wherein the alkyl and heteroaryl moieties are independently optionally substituted.

  Heteroaryl—As used herein, the term “heteroaryl” used alone or as part of a larger moiety, eg, “heteroaralkyl” or “heteroaralkoxy” With ring atoms, preferably 5, 6 or 9 ring atoms; with 6, 10 or 14 π atoms shared within the cyclic array; 1-5 in addition to carbon atoms An optionally substituted group having the heteroatoms of Heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, plinyl, naphthyridinyl, And pteridinyl. Also, the terms “heteroaryl” and “heteroar-” as used herein, a heteroaromatic ring is fused to one or more aryl, carbocyclic or heterocyclic rings, Groups in which the linking group or point of attachment is on the heteroaromatic ring are included. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolidinyl, carbazolyl, acridinyl, acridinyl, And phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, and tetrahydroisoquinolinyl. A heteroaryl group may be monocyclic or bicyclic. The term “heteroaryl” is used interchangeably with the terms “heteroaryl ring”, “heteroaryl group”, or “heteroaromatic” (both of which terms include rings that are optionally substituted). Sometimes.

  Heteroatom—As used herein, the term “heteroatom” refers to nitrogen, oxygen or sulfur and includes any oxidized form of nitrogen or sulfur and any quaternized form of basic nitrogen. The term “nitrogen” also includes substituted nitrogen.

  Heterocyclic—As used herein, the terms “heterocycle”, “heterocyclyl”, “heterocyclic group”, and “heterocyclic ring” are used interchangeably and are optionally substituted. A stable 5-7 membered monocyclic or 7-10 membered bicyclic heterocyclic moiety, either saturated or partially unsaturated, in addition to one or more carbon atoms Those having a heteroatom as defined above. A heterocyclic ring may be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure, and any ring atom may be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic groups include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl. Nyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms “heterocycle”, “heterocyclyl”, “heterocyclyl ring”, “heterocyclic group”, “heterocyclic moiety”, and “heterocyclic group” are used interchangeably herein, and The heterocyclyl ring is fused to one or more aryl, heteroaryl or carbocyclic rings (such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl or tetrahydroquinolinyl) and Includes groups where the point is on the heterocyclyl ring. The heterocyclyl group may be monocyclic or bicyclic. The term “heterocyclylalkyl” refers to an alkyl group substituted with a heterocyclyl, wherein the alkyl and heterocyclyl moieties are independently optionally substituted.

  Unsaturated—As used herein, the term “unsaturated” means that a moiety has one or more double bonds or triple bonds.

  Partially unsaturated—As used herein, the term “partially unsaturated” refers to a ring moiety that includes at least one double or triple bond. The term “partially unsaturated” is intended to include rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties as defined herein.

  Optionally substituted (optionally substituted) —As described herein, compounds of the invention may include “optionally substituted” moieties. In general, the term “substituted” means that one or more hydrogens in the indicated moiety has been replaced with a suitable substituent, with or without the term “optionally” preceding. To do. Unless otherwise stated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, from one in any given structure. If many positions can be substituted with more than one substituent selected from the specified group, the substituents can be either the same or different at each position. . Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable”, as used herein, allows for generation, detection, and in some specific embodiments, recovery, purification, and use for one or more of the purposes disclosed herein. A compound that is not substantially modified when subjected to the conditions.

Suitable monovalent substituents on the substitutable carbon atom of an “optionally substituted” group are independently halogen; — (CH ₂ ) _0-4 R °; — (CH ₂ ) _{0 -4} OR °; -O- (CH ₂ ) _0-4 C (O) OR °;-(CH ₂ ) _0-4 CH (OR ^o ) ₂ ;-(CH ₂ ) _0-4 SR °; CH _{2) 0-4} Ph (optionally substituted with _{R °) ;-( CH 2) 0-4} O (CH 2) may be substituted with 0-1 Ph (R °); - CH = CHPh (optionally substituted with R °); -NO ₂ ; -CN; -N ₃ ;-(CH ₂ ) _0-4 N (R °) ₂ ;-(CH ₂ ) _0-4 N ( -N (R °) C (S) R °;-(CH ₂ ) _0-4 N (R °) C (O) NR ° ₂ ; -N (R °) _{C (S) NR ° 2 ;-(} CH 2) 0 ^{_{4 N (R o) C (}} O) OR o; -N (R °) N (R °) C (O) R °; -N (R °) N (R °) C (O) NR ° 2; -N (R °) N (R °) C (O) OR °;-(CH ₂ ) _0-4 C (O) R °; -C (S) R °;-(CH ₂ ) _0-4 C — (CH ₂ ) _0-4 C (O) SR °; _— (CH ₂ ) _0-4 C (O) OSiR ° ₃ ; _— (CH ₂ ) _0-4 OC (O) R _{°; -OC (O) (CH} 2) 0-4 SR, -SC (S) SR ° ;-( CH 2) 0-4 SC (O) R ° ;-( CH 2) 0-4 C (O ) NR ° ₂ ; -C (S) NR ° ₂ ; -C (S) SR °; -SC (S) SR °,-(CH ₂ ) _0-4 OC (O) NR ° ₂ ; -C (O ) N (OR °) R ° ; -C (O) C (O) R °; -C (O) CH 2 C (O) R °; -C (NOR ° -(CH ₂ ) _0-4 SSR °;-(CH ₂ ) _0-4 S (O) ₂ R °;-(CH ₂ ) _0-4 S (O) ₂ OR °;-(CH ₂ ) _0-4 OS (O) ₂ R °; -S (O) ₂ NR ° ₂ ;-(CH ₂ ) _0-4 S (O) R °; -N (R °) S (O) ₂ NR _{° 2; -N (R °)} S (O) 2 R °; -N (OR °) R °; -C (NH) NR ° 2 ;-P (O) 2 R ° ;-P (O) R _{° 2; -OP (O) R} ° 2; -OP (O) (oR °) 2; SiR ° 3 ;-( C 1-4 straight or branched _{alkylene) O-N (R °)} 2 _Or- (C _1-4 straight or branched alkylene) C (O) ON (R °) ₂ , wherein each R ° is substituted as defined below Independently, hydrogen, C _1-6 aliphatic, —CH ₂ Ph, — O (CH ₂ ) _0-1 Ph, or a 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, or Regardless of the above definition, 0 to 4 independently selected two R ° are independently selected from nitrogen, oxygen or sulfur, together with their intervening atom (s) And forms a 3-12 membered saturated, partially unsaturated or aryl monocyclic or bicyclic ring, which may be substituted as defined below.

Suitable monovalent substituents on R ° (or a ring formed by two independently present R ° with its intervening atoms) are independently halogen, — (CH ₂ ) _{0. -2} R ^●, - (halo ^{_{R ●), - (CH 2}} ) 0-2 OH, - (CH 2) 0-2 OR ●, - (CH 2) 0-2 CH (OR ●) 2; -O (Halo R ^● ), —CN, —N ₃ , — (CH ₂ ) _0-2 C (O) R ^● , — (CH ₂ ) _0-2 C (O) OH, — (CH ₂ ) _0-2 C (O) OR ^● , — (CH ₂ ) _0-2 SR ^● , — (CH ₂ ) _0-2 SH, — (CH ₂ ) _0-2 NH ₂ , — (CH ₂ ) _0-2 NHR ^● , ^{_{- (CH 2) 0-2 NR ●}} 2, -NO 2, -SiR ● 3, -OSiR ● 3, -C (O) SR ●, - (C 1-4 straight or branched al Ren) C (O) OR ^●, or -SSR a ^●, wherein each R ^● are if there is a "halo" is unsubstituted, or before being substituted only with one or more halogens Independently having C _1-4 aliphatic, —CH ₂ Ph, —0 (CH ₂ ) _0-1 Ph, or independently having 0 to 4 heteroatoms selected from nitrogen, oxygen or sulfur. Selected from 5-6 membered saturated, partially unsaturated or aryl rings. Suitable divalent substituents on a saturated carbon atom at R ° include = 0 and = S.

Suitable divalent substituents on the saturated carbon atom of an “optionally substituted” group include the following: = 0, = S, = NNR ^* ₂ , = NNHC (O) R ^* , = _{^{NNHC (O) 0R *, =}} NNHS (O) 2 R *, = NR *, = N0R *, -O (C (R * 2)) 2-3 0-, or -S ^(C _{(R *} 2) ) _2-3 S-, where each independently present R ^* is hydrogen, C _1-6 aliphatic optionally substituted as defined below, or independently nitrogen, Selected from unsubstituted 5-6 membered saturated, partially unsaturated or aryl rings having 0-4 heteroatoms selected from oxygen or sulfur. Suitable divalent substituents attached to the substitutable vicinal carbon of an “optionally substituted” group include: —0 (CR ^* ₂ ) _2-3 O— (independently present) Each R ^* is hydrogen, C _1-6 aliphatic optionally substituted as defined below, or non-having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur Selected from substituted 5-6 membered saturated, partially unsaturated or aryl rings).

Suitable substituents on the aliphatic group of R ^* are halogen, —R ^● , — (halo R ^● ), —OH, —OR ^● , —O (halo R ^● ), —CN, —C (O). OH, —C (O) OR ^● , —NH ₂ , —NHR ^● , —NR ^● ₂ , or —NO ₂ (where each R ^● is unsubstituted or preceded by “halo”) Is substituted only with one or more halogens, independently from C _1-4 aliphatic, —CH ₂ Ph, —O (CH ₂ ) _0-1 Ph, or independently from nitrogen, oxygen or sulfur 5-6 membered saturated, partially unsaturated or aryl rings having 0-4 heteroatoms selected).

Suitable substituents on the substitutable nitrogen of the “optionally substituted” group include —R ^† , NR ^† ₂ , —C (O) R ^† , —C (O) OR ^† , —C (O) C (O) R †, -C (O) CH 2 C (O) R †, -S (O) 2 R †, -S (O) 2 NR † 2, -C (S) NR † ₂ , —C (NH) NR ^† ₂ , or —N (R ^† ) S (0) ₂ R ^† ; wherein each R ^† is independently hydrogen, substituted as defined below. Optionally substituted C _1-6 aliphatic, unsubstituted —OPh, or unsubstituted 5 to 6 membered saturation having 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur, or partially unsaturated or aryl ring, or, notwithstanding the definition above, independently two R ^† is present, their intervening atom (1 Tsumoshi Is an unsubstituted 3-12 membered saturated, partially unsaturated or aryl monocyclic having 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur A bicyclic ring is formed.

Suitable substituents on the aliphatic group of R ^† are independently halogen, —R ^● , — (halo R ^● ), —OH, —OR ^● , —O (halo R ^● ), —CN, — C (O) OH, —C (O) OR ^● , —NH ₂ , —NHR ^● , —NR ^● ₂ , or —NO ₂ , wherein each R ^● is unsubstituted or is ”Is substituted only with one or more halogens and is independently C _1-4 aliphatic, —CH ₂ Ph, —O (CH ₂ ) _0-1 Ph, or independently nitrogen, 5-6 membered saturated, partially unsaturated or aryl rings having 0-4 heteroatoms selected from oxygen or sulfur).

  Suitable Protecting Group—As used herein, the term “suitable protecting group” refers to an amino protecting group or a hydroxyl protecting group, depending on the position within the compound, and is described in Protecting Groups in Organic Synthesis, T.W. W. Greene and P.M. G. M.M. Those described in detail in Wuts, 3rd edition, John Wiley & Sons, 1999.

  Suitable amino protecting groups include methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9- (2-sulfo) fluorenylmethyl carbamate, 9- (2,7-dibromo) fluoroenylmethyl. Carbamate, 2,7-di-t-butyl- [9- (10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)] methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1- (1-adamantyl) -1-methylethyl carbamate (Adpoc) ), 1,1-dimethyl-2-ha Ethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1- ( 4-biphenyl) ethyl carbamate (Bpoc), 1- (3,5-di-t-butylphenyl) -1-methylethyl carbamate (t-Bumeoc), 2- (2′- and 4′-pyridyl) ethyl carbamate (Pyoc), 2- (N, N-dicyclohexylcarboxamido) ethyl carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropyl allyl carbamate (Ipaoc), cinnamylcarbame (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p- Nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinyl benzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate 2-methylsulfonylethyl carbamate, 2- (p-toluenesulfonyl) ethyl carbamate, [2- (1,3-dithianyl)] methyl carbamate (Dmoc), 4-methylthiophenylcarbamate (Mtpc), 2,4-dimethylthiophenylcarbamate (Bmpc), 2-phosphonioethylcarbamate (Peoc), 2-triphenylphosphonioisopropylcarbamate (Ppoc), 1,1-dimethyl-2 -Cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p- (dihydroxyboryl) benzyl carbamate, 5-benzisoxazolyl methyl carbamate, 2- (trifluoromethyl) -6-chromonyl methyl carbamate (Tcroc) , M-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl (o-nitrophenyl) Tilcarbamate, phenothiazinyl- (10) -carbonyl derivative, N′-p-toluenesulfonylaminocarbonyl derivative, N′-phenylaminothiocarbonyl derivative, t-amylcarbamate, S-benzylthiocarbamate, p-cyanobenzylcarbamate, Cyclobutylcarbamate, cyclohexylcarbamate, cyclopentylcarbamate, cyclopropylmethylcarbamate, p-decyloxybenzylcarbamate, 2,2-dimethoxycarbonylvinylcarbamate, o- (N, N-dimethylcarboxamido) benzylcarbamate, 1,1-dimethyl- 3- (N, N-dimethylcarboxamido) propyl carbamate, 1,1-dimethylpropynyl carbamate, di (2-pyridyl) methyl carbamate, 2 Furanylmethyl carbamate, 2-iodoethyl carbamate, isobornyl carbamate, isobutyl carbamate, isonicotinyl carbamate, p- (p′-methoxyphenylazo) benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1- (3,5-dimethoxyphenyl) ethyl carbamate, 1-methyl-1- (p-phenylazophenyl) ethyl carbamate, 1-methyl-1 -Phenylethyl carbamate, 1-methyl-1- (4-pyridyl) ethyl carbamate, phenyl carbamate, p- (phenylazo) benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate 4- (trimethylammonium) benzylcarbamate, 2,4,6-trimethylbenzyl, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridyl Carboxamide, N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N′-dithiobenzyloxycarbonylamino) acetamide, 3- (p-hydroxy Phenyl) propanamide, 3- (o-nitrophenyl) propanamide, 2-methyl-2- (o-nitrophenoxy) propanamide, 2-methyl-2- o-phenylazophenoxy) propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivative, o-nitrobenzamide, o- (benzoyloxymethyl) benzamide, 4 , 5-Diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1, 4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexane-2-one, 5-substituted 1,3-dibenzyl-1 , 3,5-triazacyclohexane-2-one, 1-substituted 3,5-dinitro-4-pyridone, N- Tylamine, N-allylamine, N- [2- (trimethylsilyl) ethoxy] methylamine (SEM), N-3-acetoxypropylamine, N- (1-isopropyl-4-nitro-2-oxo-3-pyrroline (pyroolin) ) -3-yl) amine, quaternary ammonium salt, N-benzylamine, N-di (4-methoxyphenyl) methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N -[(4-methoxyphenyl) diphenylmethyl] amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethyl Amino (Fcm), N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamino N-benzylideneamine, Np-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl) mesityl] methyleneamine, N- (N ′, N′-dimethylaminomethylene) amine, N , N′-isopropylidenediamine, Np-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N- (5-chloro-2-hydroxyphenyl) phenylmethyleneamine, N -Cyclohexylideneamine, N- (5,5-dimethyl-3-oxo-1-cyclohexenyl) amine, N-borane derivative, N-diphenylborinic acid derivative, N- [phenyl (pentacarbonylchromium- or tungsten) Carbonyl] amine, N-copper chelate, N-zinc chelate, N-nitroamine, N- Trosoamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphineamide (Mpt), diphenylthiophosphineamide (Ppt), dialkyl phosphoramidate, dibenzyl phosphoramidate, diphenyl phosphoramidate Date, benzenesulfenamide, o-nitrobenzenesulfenamide (Νps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfen Amides, 3-nitropyridinesulfenamide (Νpys), p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6, -trimethyl-4-methoxyben Sulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4 -Methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2 , 2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4- (4 ′, 8′-dimethoxynaphthylmethyl) benzenesulfonamide (DΝMBS), ben Le sulfonamide, trifluoromethyl sulfonamide, as well as Fen acylsulfonamide like.

  Suitable hydroxyl protecting groups include methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl) methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyl. Oxymethyl (PMBM), (4-methoxyphenoxy) methyl (p-A0M), guaiacol methyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM) 2,2,2-trichloroethoxymethyl, bis (2-chloroethoxy) methyl, 2- (trimethylsilyl) ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-Met Cicyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S, S-dioxide, 1-[(2-chloro-4-methyl) phenyl] -4 -Methoxypiperidin-4-yl (CTMP), 1,4-dioxane-2-yl, tetrahydrofuranyl, tetrahydrofuranyl, 2,3,3a, 4,5,6,7,7a-octahydro-7,8 , 8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1- (2-chloroethoxy) ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilyl Til, 2- (phenylselenyl) ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitro Benzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxide, diphenylmethyl, p, p′-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di (p-methoxyphenyl) phenylmethyl, tri (p-methoxyphenyl) Methyl, 4- (4′-bromophenacyloxyphenyl) di Enylmethyl, 4,4 ′, 4 ″ -tris (4,5-dichlorophthalimidophenyl) methyl, 4,4 ′, 4 ″ -tris (levulinoyloxyphenyl) methyl, 4,4 ′, 4 ″ -tris ( Benzoyloxyphenyl) methyl, 3- (imidazol-1-yl) bis (4 ′, 4 ″ -dimethoxyphenyl) methyl, 1,1-bis (4-methoxyphenyl) -1′-pyrenylmethyl, 9-anthryl, 9 -(9-phenyl) xanthenyl, 9- (9-phenyl-10-oxo) anthryl, 1,3-benzodithiolan-2-yl, benzisothiazolyl S, S-dioxide, trimethylsilyl (TMS), triethylsilyl ( TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilane DEIPS, dimethyl texylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t -Butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropi Onate, 4-oxopentanoate (levulinate), 4,4- (ethylenedithio) pentanoate (levulinoyl dithioacetal), pivaloate Adamantate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9-fluorenyl methyl carbonate (Fmoc), alkyl ethyl carbonate, Alkyl 2,2,2-trichloroethyl carbonate (Troc), 2- (trimethylsilyl) ethyl carbonate (TMSEC), 2- (phenylsulfonyl) ethyl carbonate (Psec), 2- (triphenylphosphonio) ethyl carbonate (Peoc) Alkyl isobutyl carbonate, alkyl vinyl carbonate, alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-me Xylbenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzylthiocarbonate, 4-ethoxy-1-naphthyl carbonate, methyl dithiocarbonate, 2-iodo Benzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o- (dibromomethyl) benzoate, 2-formylbenzenesulfonate, 2- (methylthiomethoxy) ethyl, 4- (methylthiomethoxy) butyrate, 2 -(Methylthiomethoxymethyl) benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4- (1,1,3,3-tetramethylbutyl) phenoxyacetate, 2 4-bis (1,1-dimethylpropyl) phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinate, (E) -2-methyl-2-butenoate, o- (methoxycarbonyl) benzoate, α-naphthoate, knight Rate, alkyl N, N, N ′, N′-tetramethyl phosphorodiamidate, alkyl N-phenyl carbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenyl sulfinate, sulfate, methanesulfonate ( Mesylate), benzyl sulfonate, and tosylate (Ts). In order to protect 1,2- or 1,3-diol, methylene acetal, ethylidene acetal, 1-tert-butyl ethylidene ketal, 1-phenyl ethylidene ketal, (4-methoxyphenyl) ethylidene acetal, 2 , 2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal, p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal, 3,4-dimethoxybenzylidene Acetal, 2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene ortho ester, 1-methoxyethylidene ortho ester, 1-eth Siethyridine orthoester, 1,2-dimethoxyethylidene orthoester, α-methoxybenzylidene orthoester, 1- (N, N-dimethylamino) ethylidene derivative, α- (N, N′-dimethylamino) benzylidene derivative, 2 -Oxacyclopentylidene orthoester, di-t-butylsilylene group (DTBS), 1,3- (1,1,3,3-tetraisopropyldisiloxanilidene) derivative (TIPDS), tetra-t-butoxydi Examples include siloxane-1,3-diylidene derivatives (TBDS), cyclic carbonates, cyclic boronates, ethyl boronates, and phenyl boronates.

  Aggregated—When two or more cells are “aggregated” by a cross-linking agent described herein, the cells are each physically associated with the cross-linking agent in a cell-drug-cell complex state. is doing. Typically, agglomeration occurs only once the crosslinker concentration reaches a threshold concentration. This concentration is referred to as the minimum aggregation concentration (MAC). The MAC of a given crosslinker is generally measured using a spectrophotometric plate reader that can quantify the change in absorbance in solution.

  Aptamer—As used herein, the term “aptamer” refers to a polynucleotide or polypeptide that specifically binds to a target molecule. In general, aptamers associate with target molecules at a detectable level, but detect similar molecular entities (eg, molecules that do not share common structural features with the target molecule) under similar conditions. If it does not associate as possible, it is said to “specifically bind” to the target molecule. The specific association between the target molecule and the aptamer typically depends on the presence or absence of certain structural features of the target molecule, such as the epitope recognized by the aptamer. In general, when an aptamer is specific for epitope A, the presence of an epitope A-containing molecule or the presence of free unlabeled epitope A in a reaction solution containing both a free labeled epitope A and an aptamer to the aptamer The amount of labeled epitope A that binds to is reduced. It will be appreciated that in general the specificity need not be absolute. Indeed, it is well known in the art that aptamers can cross-react with other epitopes in addition to the target epitope. Such cross-reactivity may be acceptable depending on the application in which the aptamer is used. Thus, the degree of aptamer specificity depends on the circumstances in which it is used. It will also be appreciated that specificity can be assessed in the context of additional factors such as the affinity of the aptamer for non-target molecules versus the affinity of the aptamer for target molecules.

Associating—As used herein, two entities are physically “associated” with each other when they are linked by direct, non-covalent interactions. Desirable non-covalent interactions include those that occur between an immunoglobulin molecule and an antigen specific for the immunoglobulin, such as ionic interactions, hydrogen bonds, van der Waals interactions, hydrophobicity Sexual interaction and the like. The strength or affinity of the physical association can be expressed in terms of the dissociation constant (K _d ) of the interaction, where the smaller the K _d , the greater the affinity. For example, the association characteristics of the selected crosslinker and target molecule can be quantified using methods well known in the art.

  Biodegradable—As used herein, the term “biodegradable” is degraded under physiological or endosomal conditions (ie, at least a portion of its covalent structure is lost). A molecule. Biodegradable molecules do not necessarily have to be hydrolytically degradable, and may require enzymatic action for degradation.

  Biomolecules—As used herein, the term “biomolecule” can be intracellular, whether naturally occurring or artificially generated (eg, synthetic or recombinant methods). And molecules commonly found in tissues (eg, polypeptides, amino acids, polynucleotides, nucleotides, polysaccharides, saccharides, lipids, nucleoproteins, glycoproteins, lipoproteins, steroids, metabolites, etc.). Specific types of biomolecules include, but are not limited to, enzymes, receptors, neurotransmitters, hormones, cytokines, cell response modifiers (such as growth factors and chemotactic factors), antibodies, vaccines, haptens, toxins , Interferons, ribozymes, antisense agents, plasmids, DNA, and RNA.

  Drug—As used herein, the term “drug” refers to a small or biomolecule that alters, inhibits, activates or affects a biological event. For example, the drugs include, but are not limited to, anti-AIDS substances, anticancer substances, antibiotics, antidiabetic substances, immunosuppressants, antiviral substances, enzyme inhibitors, neurotoxins, opioids, hypnotics, antihistamines, lubrication Drugs, tranquilizers, antispasmodics, muscle relaxants and anti-parkinsonian substances, antispasmodics and muscle contractors (such as channel blockers, miotics and anticholinergics), antiglaucoma compounds, Antiparasitic and / or antiprotozoal compounds, modulators of cell-extracellular matrix interactions (such as cell growth inhibitors and anti-adhesion molecules), vasodilators, DNA, RNA or protein synthesis inhibitors, antihypertensives, Analgesics, antipyretics, steroidal and nonsteroidal anti-inflammatory agents, antiangiogenic factors, antisecretory factors, anticoagulants Preliminary / or antithrombotic agents, local anesthetics, ophthalmic agents, prostaglandins, anti-depressants, anti-psychotic substances, anti-emetics, and imaging agents may be mentioned. A more complete listing of exemplary drugs suitable for use in the present invention can be found in “Pharmaceutical Substances: Syntheses, Patents, Applications” (by Axle Kleemann and Jurgen Engel, Inc., Thime Medical Inc .; "Chemicals, Drugs, and Biologicals", edited by Susan Budavari et al., CRC Press, 1996, and United States Pharmacopia-25 / National Formula Company, United States Pharmacopoeia. n, Inc. See Publishing, Rockville MD, 2001. Preferably (although not necessarily), the drug is one that has already been deemed safe and effective for use by an appropriate government agency or agency. For example, 21 C.I. F. R. §§ Drugs for use in humans listed in 330.5, 331-361, and 440-460; F. R. All drugs for veterinary use listed in §§ 500-589 are considered acceptable for use according to the present invention.

  Exogenous—As used herein, an “exogenous” molecule is one that is not present in a patient at a significant level unless administered to the patient. In some specific embodiments, the patient is a human. As used herein, a molecule is not present in a patient at a significant level if the molecule contained in normal human serum is less than 0.1 mM. In some specific embodiments, the molecule contained in normal human serum can be less than 0.08 mM, less than 0.06 mM, or less than 0.04 mM.

  Hyperbranched—As used herein, a “hyperbranched” structure is a covalent structure (eg, a dendrimer structure) that includes at least one branched branch. The hyperbranched structure may include a polymer substructure and / or a non-polymer substructure.

  Normal human serum—As used herein, “normal human serum” is human serum obtained by pooling approximately equal amounts of liquid portions of coagulated whole blood from eight or more healthy individuals. Healthy individuals are randomly selected individuals who are 18-30 years old who do not exhibit disease symptoms at the time of blood collection.

  Percent homology—As used herein, the term “percent homology” refers to the percent sequence identity between two sequences after optimal alignment as defined in this disclosure. For example, two nucleotide sequences are said to be “identical” if the sequence of nucleotides in the two sequences is the same when aligned for maximum match, as discussed below. Comparison of sequences between two nucleotide sequences typically compares the sequences of two optimally aligned sequences in a region or “comparison zone” to identify and compare regions where the sequences are similar Is done. The optimal alignment of sequences for comparison is described by Smith and Waterman, Ad. App. Math. 2: 482 (1981), by the local homology algorithm, Nedleman and Wunsch, J. Am. Mol. Biol. 48: 443 (1970) homology alignment algorithm, Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444 (1988), a similarity search method, computer implementation of such an algorithm, or visual inspection.

  Percent Sequence Identity— “Percent Sequence Identity” is determined by comparing two optimally aligned sequences in a comparison zone, where the portion of the nucleotide sequence in the comparison zone contains two sequences Additions or deletions (ie, gaps) may be included when compared to a reference sequence for optimal alignment of (which does not include additions or deletions). This ratio determines the number of positions where the same nucleotide residue is present in both sequences, obtains the number of matched positions, divides the number of matched positions by the total number of positions in the comparison area, and Calculated by multiplying by 100 to obtain the percent sequence identity. This definition of sequence identity given above is a definition that may be used by those skilled in the art. This definition itself does not require any algorithmic assistance. The algorithm does not calculate sequence identity, but only serves to facilitate optimal alignment of the sequences. From this definition, there will be only one distinct value for sequence identity between two sequences to be compared, and this value corresponds to the value obtained with optimal alignment.

  Polymer—As used herein, a “polymer” or “polymer structure” is a structure comprising a series of covalently bonded monomers. The polymer may be composed of one type of monomer or may be composed of more than one type of monomer. Thus, the term “polymer” encompasses copolymers (eg, block copolymers in which different types of monomers are present in individual populations throughout the polymer). The polymer may be linear or branched.

  Polynucleotide—As used herein, a “polynucleotide” is a polymer of nucleotides. The terms “polynucleotide”, “nucleic acid”, and “oligonucleotide” may be used interchangeably. The polymers include natural nucleosides (ie, adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (eg, 2-aminoadenosine, 2-thiothymidine, inosine, Pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 7-deaza Adenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O (6) -methylguanine, 4-acetylcytidine, 5- (carboxyhydroxymethyl) uridine, dihydrouridine, Lupusoiduridine, 1-methyladenosine, 1-methylguanosine, N6-methyladenosine, and 2-thiocytidine), chemically modified bases, biologically modified bases (eg, methylated bases), intervening <intercalated> bases, modifications Sugars (eg, 2′-fluororibose, ribose, 2′-deoxyribose, 2′-O-methylcytidine, arabinose, and hexose), or modified phosphate groups (eg, phosphorothioate and 5′-N-phosphoramidite) Bond).

  Polypeptide—As used herein, a “polypeptide” is a polymer of amino acids. The terms “polypeptide”, “protein”, “oligopeptide”, and “peptide” may be used interchangeably. Polypeptides include natural amino acids, non-natural amino acids (ie, compounds that are not naturally occurring but can be incorporated into a polypeptide chain) and / or amino acid analogs as are known in the art. possible. Also, one or more of the amino acid residues of the polypeptide may be a chemistry such as a sugar chain group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization or other modifications. May be modified by the addition of specific entities. Such modifications may include peptide cyclization, D-amino acid incorporation, and the like.

  Polysaccharide—As used herein, a “polysaccharide” is a carbohydrate polymer. The terms “polysaccharide”, “sugar chain”, and “oligosaccharide” may be used interchangeably. The polymer is a natural carbohydrate (eg, arabinose, lyxose, ribose, xylose, ribulose, xylulose, allose, altrose, galactose, glucose, gulose, idose, mannose, talose, fructose, psicose, sorbose, tagatose, mannohepts Sucrose, sedoheptulose, octulose, and sialose) and / or modified carbohydrates (eg, 2′-fluororibose, 2′-deoxyribose, and hexose). Exemplary disaccharides include sucrose, lactose, maltose, trehalose, gentiobiose, isomaltose, cordierbiose, laminaribiose, mannobiose, melibiose, nigerose, rutinose, and xylobiose.

  Small molecule—As used herein, the term “small molecule” has a relatively low molecular weight, whether naturally occurring or artificially created (eg, by chemical synthesis). A molecule. Typically, small molecules are monomers and have a molecular weight of less than about 1500 g / mol.

  Treat—As used herein, the term “treat” (or “treating”, “treated”, “treatment”, etc.) refers to a medical condition (eg, diabetes), a symptom of a medical condition (one or (E.g., multiple) (e.g., hyperglycemia), a substance of the present disclosure to a subject in need of treatment for the purpose of reducing, alleviating, modifying, improving, reversing, or affecting a predisposition to a medical condition Administration.

The chemical structures of AEG, AEM, AEBM and AETM are shown. The affinity for Con A of these saccharide-based affinity ligands increases as shown. The chemical structures of several exemplary conjugates, including TSB-C4 based conjugates, used in the examples are shown. (A) (B) Serum insulin and (O ) Blood glucose level plot. i-methyl mannose; p. Injections were administered at 120 minutes indicated by ^* . 1 is a schematic diagram of a cross-linked substance body 10 that can controllably release a conjugate 20 in response to an exogenous target molecule. FIG. The material body combines the conjugate 20 with a multivalent cross-linking agent 30 that binds non-covalently to the affinity ligand 40 of the conjugate 20, thereby cross-linking the conjugate 20 to form a cross-linked material body 10. It is prepared by. Non-covalent bonds between the multivalent crosslinker 30 and the affinity ligand 40 are competitively dissociated in the presence of excess amounts of exogenous target molecules. The structure of wild type human insulin is shown.

DETAILED DESCRIPTION OF SOME SPECIFIC EMBODIMENTS In this application, reference is made to a number of documents including patent and non-patent documents. Each of these documents is incorporated herein by reference in its entirety. In one aspect, according to the present disclosure, a cross-linked substance comprising a conjugate comprising a multivalent cross-linking agent that binds to an exogenous molecule, and two or more individual affinity ligands bound to a conjugate framework structure, The two or more affinity ligands compete with the exogenous molecule for binding to the cross-linking agent, and as a result of non-covalent interactions between the affinity ligand on different conjugates and the cross-linking agent, the substance Provided is a cross-linked material in which the conjugate is cross-linked in the body. The conjugate also includes a drug. The drug and affinity ligand may be covalently or non-covalently bound to the conjugate framework. In general, such substances are designed such that the release of the conjugate is stimulated by an increase in the local concentration of the exogenous molecule. The present disclosure also provides a method of using the substance, wherein a stimulating amount of an exogenous target molecule is administered to a patient who has previously been administered the substance of the present disclosure. In addition, the present disclosure provides a method for producing the substance body. In another aspect, the present disclosure provides exemplary cross-linked substances and exogenous target molecules.

  The cross-linking agent binds to an exogenous molecule (for example, but not limited to α-methyl-mannose, mannose, L-fucose, N-acetylglucosamine, a synthetic drug (eg, morphine), etc.) and is multivalent. The conjugate includes a conjugate framework that includes two or more individual affinity ligands that compete with exogenous molecules for binding to the cross-linking agent. When the crosslinker and conjugate are combined in the absence of exogenous molecules, a non-covalently crosslinked substance is formed. When the material is placed in the presence of a free exogenous molecule, the molecule competes for the interaction between the crosslinker and the conjugate. When free exogenous molecules exceed a certain concentration, the conjugate is released, resulting in a competitive level at which the substance begins to degrade. As a result, the conjugate is released from the material in a manner that is directly related to the local concentration of the exogenous molecule. In various embodiments, the substance does not substantially release the conjugate in normal human serum. The latter property substantially ensures that there is no uncontrolled release of the conjugate from the material in the absence of exogenous molecules. As discussed below, in various embodiments, it may be desirable to adjust the properties of the substance to release a certain amount of conjugate in normal human serum (eg, external To provide an intrinsically controlled component in addition to the component that is stimulated by the body).

Exogenous target molecule The present disclosure is not limited to any particular exogenous molecule. In this example, the inventors describe a substance body in which α-methyl-mannose is a stimulating substance. Since we have a much higher affinity (about 40 times) than endogenous glucose for lectin concanavalin A (Con A), we select a specific mannose derivative for the purposes of this invention. Due to this difference in binding affinity, we do not release the conjugate in response to endogenous glucose levels, but do release the conjugate in response to exogenous α-methyl-mannose. The body could be used (see Example 1). It will be appreciated that other exogenous glucose or mannose derivatives having a Con A binding affinity similar to (or greater than) α-methyl-mannose could be used as the exogenous target molecule of the substance of Example 1. Like. Examples of such compounds include, but are not limited to, mannose, L-fucose, bimannose, methylbimannose, ethylbimannose, trimannose, methyltrimannose, ethyltrimannose, and amino derivatives thereof. By Goldstein et al. Biol. Chem. 243: 2003-2007, 1968 and Biochemistry: 4: 876-883, 1965, review several Con A inhibitors and their relative affinities. Similarly, it will be appreciated that other exogenous carbohydrates (and derivatives thereof) can be used with cross-linking agents that recognize carbohydrates other than glucose or mannose (eg, other lectins, aptamers, etc.). Indeed, in some specific embodiments, it may be advantageous to use a crosslinker that does not bind endogenous glucose. Exemplary lectins that do not bind to glucose include those isolated from monocotyledonous plants such as Snowdrop (Galanthus nivalis), Garlic (Allium sativum), and Allium ursinum. Also, as discussed below, aptamers selected for lack of glucose binding may be used. Any such approach can reduce the risk of release stimulated by fluctuations in endogenous glucose levels. In various embodiments, this approach can be extended to avoid release in the presence of other endogenous molecules such as other metabolites such as creatinine, urea.

  While this example and the foregoing description involve a carbohydrate-linked crosslinker and an exogenous carbohydrate, it will be understood that the present invention is not limited to such systems. In fact, lectin-based systems are generally limited to exogenous carbohydrates, but aptamer-based systems can be designed to bind to many different exogenous molecules. For example, the method of the present invention creates a cross-linked substance that releases a drug conjugate that neutralizes the effect of the exogenous drug when the exogenous drug level becomes too high, for example, but not limited to Can be used to create a substance that releases naltrexone conjugates in response to high levels of opioids (eg, morphine, etc.). This latter example emphasizes that in various embodiments, the exogenous molecule may be a molecule that was not intentionally administered to the patient. Thus, the substances and methods of the present invention are useful in situations where stimulating substances are administered for the purpose of releasing conjugates from previously administered substances, but exogenous molecules (eg, drug abusers). Opioids such as morphine) may also be useful in situations where the substance is present to counteract non-prescription intake, injection or inhalation.

Conjugates Conjugates comprise two or more individual affinity ligands bound to a conjugate framework. The two or more individual affinity ligands compete for binding of the exogenous target molecule with the crosslinker. The conjugate also includes a drug. The affinity ligand and drug may be covalently or non-covalently bound to the conjugate framework.

Affinity ligand Two or more individual affinity ligands may have the same chemical structure or different chemical structures. The two or more individual affinity ligands may have the same chemical structure as the exogenous target molecule itself, or may be chemically related to the exogenous target molecule species. The only requirement is that the ligand compete for binding of the exogenous target molecule and the cross-linking agent. In some specific embodiments, the relative affinity of the conjugate and exogenous target molecule for the cross-linking agent is in the range of 1: 1 to 100: 1 (in this case, the relative affinity of 100: 1 is In an equilibrium mixture of conjugate, exogenous target molecule and crosslinker (at 37C in HEPES buffered saline at pH 7), when the concentration of exogenous target molecule is 100 times the concentration of conjugate, the crosslinker is approximately Means equimolar amounts of conjugate and exogenous target molecule). In some specific embodiments, this relative affinity ranges from 1: 1 to 50: 1, 1: 1 to 10: 1, 1: 1 to 5: 1, or 1: 1 to 2: 1. In various embodiments, it may be advantageous for the affinity ligand to have a different chemical structure than the exogenous target molecule, for example, to fine tune the relative affinity of the cross-linking agent for the conjugate and the exogenous target molecule. possible. For example, if the exogenous target molecule is α-methyl-mannose, a carbohydrate or polysaccharide can be used as one or more of the affinity ligands. Thus, in some specific embodiments, the affinity ligand is one that can compete with α-methyl-mannose for binding to a lectin, such as, but not limited to, Con A, mannan-binding lectin or MBL. In some specific embodiments, the affinity ligand is of formula (IVa) or (IVb):

(Where:
Each R ¹ is independently hydrogen, —OR ^y , —N (R ^y ) ₂ , —SR ^y , —O—Y, —G—Z, or —CH ₂ R ^x ;
Each R ^x is independently hydrogen, —OR ^y , —N (R ^y ) ₂ , —SR ^y , or —O—Y;
Each R ^y is independently —R ² , —SO ₂ R ² , —S (O) R ² , —P (O) (OR ² ) ₂ , —C (O) R ² , —CO ₂ R. is ² or ^{-C (O) N (R 2} ) 2,;
Each Y is independently a monosaccharide, disaccharide or trisaccharide;
Each G is independently a covalent bond or an optionally substituted C _1-9 alkylene, wherein one or more methylene units of G are optionally -O- , —S—, —N (R ² ) —, —C (O) —, —OC (O) —, —C (O) O—, —C (O) N (R ² ) —, —N ( ^{R 2) C (O) -} , - N (R 2) C (O) N (R 2) -, - SO 2 -, - SO 2 N (R 2) -, - N (R 2) SO 2 - , or _{^{-N (R 2) SO 2 N}} (R 2) - is replaced by;
Each Z is independently halogen, —N (R ² ) ₂ , —OR ² , —SR ² , —N ₃ , —C≡CR ² , —CO ₂ R ² , —C (O) R ² , or be a -OSO ₂ R ^2;
Each R ² is independently hydrogen or a 4-7 membered heterocyclic having 1-2 heteroatoms selected from C _1-6 aliphatic, phenyl, nitrogen, oxygen or sulfur Or an optionally substituted group selected from 5 to 6-membered monocyclic heteroaryl rings having 1 to 4 heteroatoms selected from nitrogen, oxygen or sulfur) belongs to.

  In some specific embodiments, the affinity ligand of formula (IVa) or (IVb) is a monosaccharide. In some specific embodiments, the affinity ligand is a disaccharide. In some specific embodiments, the affinity ligand is a trisaccharide. In some specific embodiments, the affinity ligand is a tetrasaccharide. In some specific embodiments, affinity ligands are those that contain no more than 4 carbohydrate moieties.

As generally defined above, each R ¹ is independently hydrogen, —OR ^y , —N (R ^y ) ₂ , —SR ^y , —O—Y, —G—Z, or —CH. ₂ R ^x . In some specific embodiments, R ¹ is hydrogen. In some specific embodiments, R ¹ is —OH. In other embodiments, R ¹ is —NHC (O) CH ₃ . In some specific embodiments, R ¹ is —O—Y. In some other other embodiments, R ¹ is —GZ. In some embodiments, R ¹ is —CH ₂ OH. In other embodiments, R ¹ is —CH ₂ —O—Y. In yet other embodiments, R ¹ is —NH ₂ . ^One skilled in the art will recognize that each R ¹ substituent in formula (IVa) or (IVb) can be the stereochemical structure of (R) or (S).

As defined generally above, each R ^x is independently hydrogen, −0R ^y , —N (R ^y ) ₂ , —SR ^y , or —O—Y. In some embodiments, R ^x is hydrogen. In some specific embodiments, R ^x is —OH. In other embodiments, R ^x is —O—Y.

As generally defined above, each R ^y is independently —R ² , —SO ₂ R ² , —S (O) R ² , —P (O) (OR ² ) ₂ , —C. (O) R ² , —CO ₂ R ² , or —C (O) N (R ² ) ₂ . In some embodiments, R ^y is hydrogen. In other embodiments, R ^y is —R ² . In some embodiments, R ^y is —C (O) R ² . In some specific embodiments, R ^y is acetyl. In other embodiments, R ^y is —SO ₂ R ² , —S (O) R ² , —P (O) (OR ² ) ₂ , —CO ₂ R ² , or —C (O) N (R ² ) It is ₂ .

  As generally defined above, Y is a monosaccharide, disaccharide or trisaccharide. In some specific embodiments, Y is a monosaccharide. In some embodiments, Y is a disaccharide. In other embodiments, Y is a trisaccharide. In some embodiments, Y is mannose, glucose, fructose, galactose, rhamnose, or xylopyranose. In some embodiments, Y is sucrose, maltose, tunulose, trehalose, cellobiose, or lactose. In some specific embodiments, Y is mannose. In some specific embodiments, Y is D-mannose. One skilled in the art will recognize that sugar Y is attached to the oxygen group of —O—Y via the anomeric carbon to form a glycosidic bond. The glycosidic bond may be in the α configuration or the β configuration.

As defined generally above, each G is independently a C _1-9 alkylene that is a covalent bond or is optionally substituted, wherein one or more methylenes of G units, ^{optionally, -0 -, - S -,} - N (R 2) -, - C (O) -, - OC (O) -, - C (O) O -, - C (O) N (R ² ) —, —N (R ² ) C (O) —, —N (R ² ) C (O) N (R ² ) —, —SO ₂ —, —SO ₂ N (R ² ) — , —N (R ² ) SO ₂ — or —N (R ² ) SO ₂ N (R ² ) —. In some embodiments, G is a double bond. In some specific embodiments, G is -0-C _1-8 alkylene. In some specific embodiments, G is —OCH ₂ CH ₂ —.

As generally defined above, each Z is independently halogen, —N (R ² ) ₂ , —OR ² , —SR ² , —N ₃ , —C≡CR ² , —CO ₂ R. ^{2, -C (O) R 2} , or -OSO ₂ R ^2. In some embodiments, Z is halogen or —OSO ₂ R ² . In other embodiments, Z is —N ₃ or —C≡CR ² . In some specific embodiments, Z is —N (R ² ) ₂ , —OR ² , or —SR ² . In some specific embodiments, Z is —SH. In some specific embodiments, Z is —NH ₂ . In certain embodiments, -G-Z is -OCH ₂ CH ₂ NH _2.

In some embodiments, the R ¹ substituent on the C1 carbon of formula (IVa) is —GZ, and formula (IVa-i):

Where R ¹ , G and Z are as defined and described herein.

In some embodiments, the ligand is of formula (IVa-ii):

Wherein R ¹ , R ^x , G and Z are as defined and described herein.

For example, in some specific embodiments, the following: glucose, sucrose, maltose, mannose, derivatives thereof (eg, glucosamine, mannosamine, methyl glucose, methyl mannose, ethyl glucose, ethyl mannose, etc.) and / or higher Affinity ligands comprising one or more of the following combinations (eg, bimannose, linear and / or branched trimannose, etc.) may be used. In some specific embodiments, the affinity ligand comprises a monosaccharide. In some specific embodiments, the affinity ligand comprises a disaccharide. In some specific embodiments, the affinity ligand comprises a trisaccharide. In some specific embodiments, the affinity ligand comprises a polysaccharide. In some embodiments, the affinity ligand comprises a sugar and one or more amine groups. In some embodiments, the affinity ligand is aminoethyl glucose (AEG). In some embodiments, the affinity ligand is aminoethyl mannose (AEM). In some embodiments, the affinity ligand is aminoethylbimannose (AEBM). In some embodiments, the affinity ligand is aminoethyltrimannose (AETM). In some embodiments, the affinity ligand is β-aminoethyl-N-acetylglucosamine (AEGA). In some embodiments, the affinity ligand is aminoethyl fucose (AEF). In other embodiments, the affinity ligand is D-glucosamine (GA). In some specific embodiments, the sugar ligand is of the “D” configuration. In other embodiments, the sugar ligand is of the “L” configuration. In the following, we show the structures of these exemplary affinity ligands. Other exemplary affinity ligands will be recognized by those skilled in the art.

  In various embodiments, the affinity ligand is a polysaccharide, glycopeptide or glycolipid. In some specific embodiments, the affinity ligand comprises 2-10 carbohydrate moieties, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 such moieties. The terminal and / or internal residues of a polysaccharide, glycopeptide or glycolipid can be selected based on the sugar specificity of the lectin of interest (eg, Goldstein et al., Biochem. Biophys. Acta 317: 500-504, 1973 and Lis). Et al., Ann. Rev. Biochem. 55: 35-67, 1986).

  In various embodiments, the affinity ligand for a particular conjugate / crosslinker combination can be selected based on experimentation. According to such embodiments, one or more affinity ligands are screened based on the relative binding affinity for the crosslinker as compared to the exogenous target molecule (and possibly the endogenous molecule discussed below). Is done. In some specific embodiments, a library of carbohydrates and / or polysaccharides is screened in this manner. Suitable affinity ligands are those that exhibit a detectable level of competition with the exogenous target molecule, but do not compete so strongly as to completely prevent binding between the crosslinker and the exogenous target molecule.

  In various embodiments, the affinity ligand is selected based on its ability to compete with the endogenous molecule for the cross-linking agent. In particular, in certain embodiments, it has a much higher affinity for the cross-linking agent than a potential competitive endogenous molecule (eg, glucose if the cross-linking agent is glucose-binding). It may be advantageous to select an affinity ligand that cannot competitively bind to a suitable exogenous target molecule (eg, α-methyl-mannose). This minimizes the degree of release from the cross-linked material in the absence of exogenous target molecules. For example, in Examples 1-2, we showed little background release in the presence of physiological levels of endogenous glucose, but did not respond significantly to exogenous α-methyl-mannose. Illustrative conjugates with two resulting AEBM affinity ligands are described. In other embodiments, a certain amount of glucose-responsive release is possible, but it may be desirable to select an affinity ligand that can be exogenously stimulated to artificially modulate the release.

  Other exemplary exogenous target molecule / affinity ligand combinations will be recognized by those skilled in the art. In general, affinity ligands were obtained using the target molecule itself and / or by making a derivative of the target molecule (eg, making chemical and / or stereochemical modifications to the target molecule and then obtaining Derivatives can be made against any exogenous target molecule (by screening for relative affinity for the subject cross-linking agent).

  As discussed in more detail below, affinity ligands may be naturally occurring within the framework of the conjugate (eg, as part of the polymer backbone or as a side chain group of monomers). ). Alternatively (or additionally), the affinity ligand may be artificially incorporated into the conjugate framework (eg, synthetically added to the conjugate framework). In the form of chemical groups). In some specific embodiments, the conjugate comprises a framework that includes 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, or 100 or more affinity ligands. obtain. In some specific embodiments, the conjugate comprises a framework structure comprising 2-5, 2-10, 2-20, 2-25, 2-50, or 2-100 affinity ligands. obtain. In some specific embodiments, the conjugate may comprise a framework structure that includes as few as 2, 3 or 4 individual affinity ligands.

  Methods for conjugating affinity ligands to the conjugate framework are discussed in more detail below. In some specific embodiments, when the affinity ligand comprises a sugar, conjugation (whether direct or indirect) involves the C1, C2 or C6 position of the sugar. In some specific embodiments, the conjugation involves the C1 position. The C1 position, also referred to as the anomeric carbon, can be linked to the framework of the conjugate in an α or β conformation. In some specific embodiments, the C1 position is configured as an α anomer. In other embodiments, the C1 position is configured as a β anomer.

Drug As noted above, the conjugate also includes a drug. It will be appreciated that the conjugate can include any drug. Conjugates can contain more than one copy of the same drug and / or can contain more than one type of drug. Conjugates are not limited to any particular drug, and may include small molecule drugs or biomolecule drugs. In general, the drug (s) used depends on the disease or disorder being treated.

  For example, but not limited to, in various embodiments, the conjugate comprises the following drug: diclofenac, nifedipine, rivastigmine, methylphenidate, fluoxetine, rosiglitazone, prednisone, prednisolone, codeine, ethylmorphine, dextromethorphan, noscapine, Pentoxiverine, acetylcysteine, bromhexine, epinephrine, isoprenaline, orciprenaline, ephedrine, fenoterol, limiterol, ipratropium, choline theophyllate, proxyphylline, beclomethasone, budesonide, deslanoside, digoxin, sodigitoximine Quinidine, procainamide, mexiletine, flecainide, alp Nolol, propranolol, nadolol, pindolol, oxprenolol, labetalol, timolol, atenolol, pentaerythritol tetranitrate, isosorbide nitrate, isosorbide mononitrate, nifedipine, phenylamine, verapamil, diltiazem, cyclandelil (cyclinderil) Alcohol, inositol nicotinate, alprostatdil, ethylephrine, prenalterol, dobutamine, dopamine, dihydroergotamine, guanethidine, betanidine, methyldopa, reserpine, guanfacine, trimetaphane, hydralazine, dihydralazine, prazosin, peptoxol, diazoloxide Nifedipine, enalapril Nitroprusside, bendroflumethiazide, hydrochlorothiazide, methycrothiazide, polythiazide, chlorthalidone, cinetazone, clopamide, mefluside, metolazone, bumetanide, ethacrinaside, spironolactone, amiloride, clofibrate, nicotinic acid, nicheliritrol, nicoritolol Dexicyclopheniramine, clemastine, antazoline, cyproheptadine, promethazine, cimetidine, ranitidine, sucralfate, papaverine, moxaverine, atropine, butyl scopolamine, emeprone, glucopyrone, hyoscyamine, mepensolarine, methocopamine, methyl scopomine , Terodiline , Senna glycoside, sagrada extract, dantron, bisacodyl, sodium picosulfate, ethuros, diphenoxylate, loperamide, salazosulfapyridine, pyruvin, mebendazole, dimethicone, ferrofumarate, ferrosuccinate, ferritetrasemi Sodium, cyanocobalamin, folate heparin, heparin cofactor, diculmarol, warfarin, streptokinase, urokinase, factor VIII, factor IX, vitamin K, thiotepa, busulfan, chlorambucil, cyclophosphamide, melphalan, carmustine, mercapto Purine, thioguanine, azathioprine, cytarabine, vinblastine, vincristine, vindesine, procarbazine, Daka Vadine, Lomustine, Estramustine, Teniposide, Etoposide, Cisplatin, Amsacrine, Aminoglutethimide, Phospherolone, Medroxyprogresterone, Hydroxyprogesterone, Megesterol, Noretisterone, Tamoxifen, Cyclosporine, Sulphisidine , Phenoxymethylpenicillin, dicloxacillin, cloxacillin, flucloxacillin, ampicillin, amoxilin, pivampicillin, bacampicillin, piperacillin, mediocillin, mecillinam, pibmesilinum, cephalothin, cephalexin, cefradixifel, cefadroxift Threonam, imipenem, cilastatin, tetracycline, limecycline, demeclocycline, metacycline, oxytetracycline, doxycycline, chloramphenicol, spiramycin, fusidic acid, lincomycin, clindamycin, spectinomycin, rifampicin, amphotericin B, Griseofulvin, nystatin, vancomycin, metronidazole, tinidazole, trimethoprim, norfloxacin, salazosulfapyridine, aminosalil, isoniazid, ethambutol, nitrofurantoin, nalidixic acid, methenamine, chloroquine, hydroxychloroquine, tinidazole, cyclotoconazole, ketoconazole Idoxuridine, retinol, Thiamine, dexpanthenol, pyridoxine, folic acid, ascorbic acid, tocopherol, phytominadione, fenflunamamine, corticotropin, tetracosactide, thyrotropin, somatotropin, somatrem, vasopressin, ripressin, desmopressin, oxytocin, chloritogonadohydrocortisone, cortisone Drocortisone, prednisone, prednisolone, fluoxymesterone, mesterolone, nandrolone, stanozolol, oxymetholone, cyproterone, levothyroxine, liothyronine, propylthiouracil, carbimazole, thiamazole, dihydrotaxosterol, alphacalcidol, calcitilol, insulin, tolbutamide, Chlorpropamide, tolazamide, glipizide Glibenclamide, phenobarbital, metiprilone, pyrityldione, meprobamate, chlordiazepoxide, diazepam, nitrazepam, baclofen, oxazepam, dipotassium chlorazepate, lorazepam, flunitrazemazine, troprazemazine Chlorpromazine, levomepromazine, acetophenazine, fluphenazine, perphenazine, prochlorpenazine, trifluoperazine, dixylazine, thioridazine, periciazine, cloprothixene, tizanidine, zalprone, zuclopentizol , Thixixene, haloperidol, trimipramine, opipramol, clomipramine, desipramine, lofepramine, amitriptyline, nortriptyline, protriptyline, maptrotilin, caffeine, cinnarizine, cyclidine, methhydrinate (dimenhydrinate), meclozine, promethazine, thiethylperazine, thiethylperazine Phenobarbital, phenytoin, ethosuximide, primidone, carbamazepine, clonazepam, orphenadrine, atropine, benzatropin, biperidene, methixene, procylidin, levodopa, bromocriptine, amantadine, ambenone, pyridostigmine, pentastigmine, disulficobine, disulficobine Ruphine, pethidine, phenoperidine, fentanyl, methadone, pyritramide, dextropropoxyphene, ketobemidon, acetylsalicylic acid, celecoxib, phenazone, phenylbutazone, azapropazone, piroxicam, ergotamine, dihydroergotamine, cyproheptadine, pidithiophene, flumedroproneneol Sodium auranofin, penicillamine, estrdiol, estradiol valerate, estriol, ethinyl estradiol, dihydrogesterone, linestrenol, medroxyprogresterone, norethisterone, cyclophenyl, clomiphene, levonorgestrel, mestranol, ornidazole , Tinidazole, Conazole, clotrimazole, levonorgestrel, mestranol, ornidazole, tinidazole, econazole, clotrimazole, natamycin, miconazole, sulbene, methylergotamine, dinoprost, dinoprostone, gemeprost, bromocriptine, phenylpropanolamine, sodium cromoglycate, acetazolamide, diclofenamide It may contain any one of beta carotene, naloxone, folinate calcium, especially clonidine, theophylline, dipyridamole, hydrochlorthiazide, scopolamine, indomethacin, furosemide, potassium chloride, morphine, ibuprofen, salbutamol, terbutaline calcitonin. It will be appreciated that this list is intended to be exemplary and that any drug, whether known or later found, can be used in the conjugates of the present disclosure.

  In various embodiments, the conjugate is a hormonal drug (which may be peptide-based or non-peptide-based) such as adrenaline, noradrenaline, angiotensin, atriopeptin, aldosterone, dehydroepiandrosterone, androsten. Dione, testosterone, dihydrotestosterone, calcitonin, calcitriol, calcidiol, corticotropin, cortisol, dopamine, estradiol, estrone, estriol, erythropoietin, follicle stimulating hormone, gastrin, ghrelin, glucagon, gonadotropin releasing hormone, growth Hormone, growth hormone releasing hormone, human chorionic gonadotropin, histamine, human placental lactogen, insulin, insulin-like growth factor, insulin , Leptin, leukotriene, lipotropin, melatonin, orexin, oxytocin, parathyroid hormone, progesterone, prolactin, prolactin releasing hormone, prostaglandin, renin, serotonin, secretin, somatostatin, thrombopoietin, thyroid stimulating hormone, thyroid stimulating hormone It may include release hormone (or thyroid stimulating hormone), thyroid stimulating hormone releasing hormone, thyroxine, triiodothyronine, vasopressin, and the like. In some specific embodiments, the hormone is glucagon, insulin, insulin-like growth factor, leptin, thyroid stimulating hormone, thyroid stimulating hormone releasing hormone (or thyroid stimulating hormone), thyroid stimulating hormone releasing hormone, thyroxine, and triiodine It can be selected from thyronine. It will be understood that this list is intended to be exemplary and that any hormonal drug, whether known or later found, can be used in the conjugates of the present disclosure.

  In various embodiments, the conjugate can include thyroid hormone.

  In various embodiments, the conjugate can include an anti-diabetic drug (ie, a drug that has a beneficial effect on a patient suffering from diabetes).

  In various embodiments, the conjugate can include an insulin molecule. By “insulin molecule” we include both wild-type and modified insulin as long as they are bioactive (ie, can cause a decrease in detectable glucose when administered in vivo). Intended. Wild type insulin includes insulin from any species, whether purified, synthetic or recombinant (eg, human insulin, porcine insulin, bovine insulin, rabbit insulin, sheep insulin, etc.) . Some of these are commercially available, for example from Sigma-Aldrich (St. Louis, MO). A variety of modified insulins are known in the art (eg, Crotty and Reynolds, Pediatr. Emerg. Care. 23: 903-905, 2007, and Gerich, Am. J. Med. 113: 308-16, 2002, and references cited therein). Modified insulins are chemically modified (eg, by addition of a chemical moiety such as a PEG group or an aliphatic acyl chain described below) and / or mutated (ie, addition, deletion of one or more amino acids). Or by substitution). In general, physiologically active mutant forms of insulin typically differ from wild-type insulin by 1-10 (eg, 1-5 or 1-2) amino acid substitutions, additions or deletions. The wild type sequence of human insulin (A chain and B chain) is shown below and in FIG.

A chain (SEQ ID NO: 1): GIVEQCTCTSICSLYQLENYCN
B chain (SEQ ID NO: 2): FVNQHLCGSHLVALELYLVCGGERGFYTPKT
Human insulin differs from rabbit, porcine, bovine, and sheep insulin only in amino acids A8, A9, A10, and B30 (see table below).

In various embodiments, an insulin molecule of the present disclosure is one in which positions B28 and / or B29 of the B peptide sequence are mutated. For example, insulin lispro (HUMALOG®) is a fast-acting insulin mutant (Lys ^B28 Pro ^{B29 -human} insulin) in which the second lysine and proline residue from the end of the C-terminal of the B peptide are reversed. This modification blocks the formation of insulin multimers. Insulin aspart (NOVOLOG®) is another fast-acting insulin variant (Asp ^{B28 -human} insulin) in which the proline at position B28 is replaced with aspartic acid. This variant also suppresses the formation of multimers. In some embodiments, the mutation at position B28 and / or B29 is accompanied by one or more mutations elsewhere in the insulin polypeptide. For example, insulin gllysine (APIDRA®) is another fast-acting insulin variant (Lys) in which aspartic acid at position B3 is replaced with a lysine residue and lysine at position B29 is replaced with a glutamic acid residue. ^B3 Glu ^{B29 -human} insulin).

In various embodiments, the insulin molecules of the present disclosure have a shifted isoelectric point compared to human insulin. In some embodiments, the isoelectric point shift is made by adding one or more arginine residues to the N-terminus of the insulin A peptide and / or the C-terminus of the insulin B peptide. Examples of such insulin polypeptides include Arg ^{A0 -human} insulin, Arg ^B31 Arg ^{B32 -human} insulin, Gly ^A21 Arg ^B31 Arg ^{B32 -human} insulin, Arg ^A0 Arg ^B31 Arg ^{B32 -human} insulin, and Arg ^A0 Gly ^A21 Arg ^B31 Arg ^{B32 -human} insulin. As a further example, insulin glargine (LANTUS®) is an exemplary long acting insulin variant in which Asp ^A21 is replaced with glycine and two arginine residues are added to the C-terminus of the B peptide. The effect of these changes is that the isoelectric point shifts and a completely soluble solution at pH 4 is obtained. Thus, in some embodiments, an insulin molecule of the present disclosure comprises an A peptide sequence in which A21 is Gly and a B peptide sequence in which B31 is Arg-Arg. The present disclosure describes these and any other mutations described herein (eg, Gly ^{A21 -human} insulin, Gly ^A21 Arg ^{B31 -human} insulin, Arg ^B31 Arg ^{B32 -human} insulin, Arg ^{B31 -human} insulin) It will be understood to encompass all of the single and many combinations of.

  In various embodiments, the insulin molecules of the present disclosure are truncated. For example, in some specific embodiments, the B peptide sequence of an insulin polypeptide of the present disclosure is one without B1, B2, B3, B26, B27, B28, B29 and / or B30. In some specific embodiments, there are no residue combinations in the B peptide sequence of the disclosed insulin polypeptides. For example, the B peptide sequence may comprise residues B (1-2), B (1-3), B (29-30), B (28-30), B (27-30) and / or B (26- 30) may be absent. In some embodiments, these deletions and / or truncations apply to any of the insulin molecules described above (eg, but not limited to des (B30) -insulin lispro, des (B30) -insulin aspart. , Des (B30) -insulin gluridine, des (B30) -insulin glargine and the like).

  In some embodiments, the insulin molecule comprises an additional amino acid residue at the N- or C-terminus of the A or B peptide sequence. In some embodiments, there are one or more amino acid residues at positions A0, A21, B0 and / or B31. In some embodiments, there is one or more amino acid residues at position A0. In some embodiments, there is one or more amino acid residues at position A21. In some embodiments, there is one or more amino acid residues at position B0. In some embodiments, one or more amino acid residues are present at position B31. In some specific embodiments, the insulin molecule does not contain any additional amino acid residues at positions A0, A21, B0 or B31.

In some specific embodiments, the insulin molecules of the present disclosure are mutated such that one or more amidated amino acids are replaced with an acidic form. For example, asparagine can be replaced with aspartic acid or glutamic acid. Similarly, glutamine can be replaced with aspartic acid or glutamic acid. In particular, Asn ^A18 , Asn ^A21 , or Asn ^B3 , or any combination of these residues can be replaced with aspartic acid or glutamic acid. Gln ^A15 or Gln ^B4 or both can be replaced with aspartic acid or glutamic acid. In some specific embodiments, the insulin molecule has aspartic acid at position A21, or aspartic acid at position B3, or both.

One skilled in the art will recognize that other amino acids within the insulin molecule can be mutated while retaining biological activity. For example, without limitation, the following modifications: replacement of the histidine residue at position B10 with aspartic acid (His ^B10 → Asp ^B1 °); replacement of the phenylalanine residue at position Bl with aspartic acid (Phe ^B1 → Asp ^B1 ); Replacement of threonine residue at position B30 with alanine (Thr ^B30 → Ala ^B30 ); Replacement of tyrosine residue at position B26 with alanine (Tyr ^B26 → Ala ^B26 ); and serine residue at position B9 with aspartic acid Replacement (Ser ^B9 → Asp ^B9 ) is also widely accepted in the art.

In various embodiments, insulin molecules of the present disclosure are those that have a sustained action profile. Thus, in some specific embodiments, insulin molecules of the present disclosure can be acylated with fatty acids. That is, an amide bond is formed between the amino group on the insulin molecule and the carboxylic acid group of the fatty acid. The amino group may be the α-amino group of the N-terminal amino acid of the insulin molecule or the ε-amino group of the lysine residue of the insulin molecule. Insulin molecules of the present disclosure may be those in which one or more of the three amino groups present in wild type insulin are acylated, or in which lysine residues introduced into the wild type sequence are acylated. possible. In some specific embodiments, the insulin molecule may be acylated at the B1 position. In some specific embodiments, the insulin molecule may be acylated at position B29. In some specific embodiments, the fatty acid is selected from myristic acid (C14), pentadecylic acid (C15), palmitic acid (C16), heptadecylic acid (C17) and stearic acid (C18). For example, insulin detemir (LEVEMIIR®) is a long-acting insulin variant in which Thr ^B30 is deleted and a C14 fatty acid chain (myristic acid) is linked to Lys ^B29 .

In some embodiments, the N-terminus of the A peptide, the N-terminus of the B peptide, the ε-amino group of Lys at position B29, or any other available amino group in the insulin molecule of the present disclosure has the general formula :

_Where R ^F is hydrogen or a C _1-30 alkyl group and is covalently bonded to the fatty acid moiety. In some embodiments, R ^F is C _1-20 alkyl group, C _3-19 alkyl group, C _5-18 alkyl group, C _6-17 alkyl group, C _8-16 alkyl group, C _10-15. It is an alkyl group or a C _12-14 alkyl group. In some specific embodiments, the insulin polypeptide is conjugated to the moiety at the A1 position. In some specific embodiments, the insulin polypeptide is conjugated to the moiety at the B1 position. In some specific embodiments, the insulin polypeptide is conjugated to the moiety at the ε-amino group of Lys at position B29. In some specific embodiments, position B28 of the insulin molecule is Lys and the ε-amino group of Lys ^B28 is conjugated to a fatty acid moiety. In some specific embodiments, the B3 position of the insulin molecule is Lys and the ε-amino group of Lys ^B3 is conjugated to a fatty acid moiety. In some embodiments, the fatty acid chain is 8-20 carbons long. In some embodiments, the fatty acid is octanoic acid (C8), nonanoic acid (C9), decanoic acid (C10), undecanoic acid (C11), dodecanoic acid (C12), or tridecanoic acid (C13). In some specific embodiments, the fatty acid is myristic acid (C14), pentadecanoic acid (C15), palmitic acid (C16), heptadecanoic acid (C17), stearic acid (C18), nonadecanoic acid (C19), or arachidin. Acid (C20). For example, insulin detemir (LEVEMIIR®) is a long-acting insulin variant in which Thr ^B30 is deleted and a C14 fatty acid chain (myristic acid) is linked to Lys ^B29 .

In some specific embodiments, an insulin molecule of the present disclosure comprises the following insulin molecules: Lys ^B28 Pro ^{B29 -human} insulin (insulin lispro), Asp ^{B28 -human} insulin (insulin aspart), Lys ^B3 Glu ^B29 -human Insulin (insulin ^gluridine ), Arg ^B31 Arg ^B32 -human insulin (insulin glargine), N [ ^{epsilon] B29} -myristoyl- ^des (B30) ^-human insulin (insulin ^detemil ), Ala ^B26 -human insulin, Asp ^B1 -human insulin, Arg ^A0 -Human insulin, Asp ^B1 Glu ^B13 -Human insulin, Gly ^A21 -Human insulin, Gly ^A21 Arg ^B31 Arg ^B32 -Human insulin, Arg ^A0 Arg ^B31 Arg ^B32 -human insulin, Arg ^A0 Gly ^A21 Arg ^B31 Arg ^B32 -human insulin, des (B30) -human insulin, des (B27) -human insulin, des (B28-B30) -human insulin, des (B1) -human insulin , Des (B1-B3) -containing one mutation and / or chemical modification of human insulin. In some specific embodiments, an insulin molecule of the present disclosure comprises the following insulin molecules: ^NεB29 -palmitoyl-human insulin, ^{NεB29-myrisoyl-} human insulin, ^NεB28 -palmitoyl-Lys ^B28 Pro ^B29 − human ^insulin, N εB28 - including one of the human insulin mutant and / or chemically modified - myristoylation ^-Lys B28 ^{Pro B29.}

In some specific embodiments, an insulin molecule of the present disclosure has the following insulin molecules: N ^{εB29 -palmitoyl-des} (B30) ^-human insulin, N ^εB30 -myristoyl-Thr ^B29 Lys ^B30 -human insulin, N ^εB30- Palmitoyl-Thr ^B29 Lys ^B30 -human insulin, ^NεB29- (N-palmitoyl-γ-glutamyl) -des (B30) ^-human insulin, ^NεB29- (N-ritocryl-γ-glutamyl) -des (B30) -human It includes one mutation and / or chemical modification of insulin, N [ ^{epsilon] B29} -([omega] -carboxyheptadecanoyl) -des (B30) ^-human insulin, N [ ^{epsilon] B29} -([omega] -carboxyheptadecanoyl) ^-human insulin.

In some specific embodiments, an insulin molecule of the present disclosure has the following insulin molecules: ^NεB29 -octanoyl-human insulin, ^NεB29 -myristoyl- ^{Gly A21} Arg ^B31 Arg ^B31 -human insulin, ^NεB29 -myristoyl- ^{Gly. A21} Gln ^B3 Arg ^B31 Arg ^B32 - human insulin, N ^{&lt; [epsilon] B29} - myristoyl ^{-Arg A0 Gly A21 Arg B31 Arg B32} - human ^{insulin, N εB29 -Arg A0 Gly A21 Gln} B3 Arg B31 Arg B32 - human insulin, N ^{&lt; [epsilon] B29} - myristoyl - ^{Arg A0 Gly A21 Asp B3 Arg B31} Arg B32 - human insulin, N ^{&lt; [epsilon] B29} - myristoyl ^-Arg B31 ^{Arg B32} - human insulin, ^{&lt; [epsilon] B29} - myristoyl ^-Arg A0 ^Arg B31 ^{Arg B32} - human insulin, N ^{&lt; [epsilon] B29} - octanoyl ^-Gly A21 ^Arg B31 ^{Arg B32} - human insulin, N ^{&lt; [epsilon] B29} - octanoyl ^{-Gly A21 Gln B3 Arg B31 Arg B32} - human insulin, N ^{&lt; [epsilon] B29} - octanoyl ^{-Arg A0 Gly A21 Arg B31 Arg B32} - human insulin, N ^{&lt; [epsilon] B29} - octanoyl ^{-Arg A0 Gly A21 Gln B3 Arg B31} Arg B32 - human insulin, N ^{&lt; [epsilon] B29} - octanoyl ^{-Arg B0 Gly A21 Asp B3 Arg B31} Arg B32 - human insulin ^NεB29 -octanoyl-Arg ^B31 Arg ^B32 -human insulin, ^NεB29 -octanoy It contains mutations and / or chemical modifications of one of the following: Lu-Arg ^A0 Arg ^B31 Arg ^{B32 -human} insulin.

In certain embodiments, the insulin molecule of the present disclosure, the following insulin ^polypeptide: N εB28 - myristoyl ^{-Gly A21 Lys B28 Pro B29 Arg B31} Arg B32 - human ^insulin, N εB28 - myristoyl ^-Gly A21 ^{Gln B3 Lys B28 Pro B30 Arg B31 Arg B32} - human ^insulin, N εB28 - myristoyl ^{-Arg A0 Gly A21 Lys B28 Pro B29} Arg B31 Arg B32 - human ^insulin, N εB28 - myristoyl ^{-Arg A0 Gly A21 Gln B3 Lys B28} Pro B29 Arg B31 arg ^B32 - human ^insulin, N εB28 - myristoyl ^{-Arg A0 Gly A21 Asp B3 Lys B28} Pro B29 Ar ^B31 Arg ^B32 - human ^insulin, N εB28 - myristoyl ^{-Lys B28 Pro B29 Arg B31 Arg B32} - human ^insulin, N εB28 - myristoyl ^{-arg A0 Lys B28 Pro B29 Arg B31} Arg B32 - human ^insulin, N εB28 - octanoyl ^{-Gly A21} Lys ^B28 Pro ^B29 Arg ^B31 Arg ^{B32—Contains} mutations and / or chemical modifications of one of the human insulins.

In certain embodiments, the insulin molecule of the present disclosure, the following insulin ^molecule: N εB28 - octanoyl ^{-Gly A21 Gln B3 Lys B28 Pro B29} Arg B31 Arg B32 - human ^insulin, N εB28 - octanoyl ^-Arg A0 Gly ^{A21 Lys B28 Pro B29 Arg B31 Arg} B32 - human ^insulin, N εB28 - octanoyl ^{-Arg A0 Gly A21 Gln B3 Lys B28} Pro B29 Arg B31 Arg B32 - human ^insulin, N εB28 - octanoyl ^{-Arg A0 Gly A21 Asp B3 Lys B28} Pro ^B29 Arg ^B31 Arg ^{B32 -human} insulin, ^NεB28 -octanoyl-Lys ^B28 Pro ^B29 Arg ^B31 Arg ^B32 - including the one of the human insulin mutant and / or chemically modified - human ^insulin, N εB28 - octanoyl ^{-Arg A0 Lys B28 Pro B29 Arg B31} Arg B32.

In some specific embodiments, an insulin molecule of the present disclosure comprises the following insulin molecules: ^NεB29 -tridecanoyl- ^des (B30) ^-human insulin, ^NεB29 -tetradecanoyl- ^des (B30) ^-human insulin, N ^{&lt; [epsilon] B29} - decanoyl -des (B30) - human insulin, N ^{&lt; [epsilon] B29} - dodecanoyl -des (B30) - human insulin, N ^{&lt; [epsilon] B29} - tridecanoyl ^-Gly A21 -des (B30) - human insulin, N ^{&lt; [epsilon] B29} - tetradecanoyl ^{-Gly A21} -des (B30) - human insulin, N ^{&lt; [epsilon] B29} - decanoyl ^-Gly A21 -des (B30) - human insulin, N ^{&lt; [epsilon] B29} - dodecanoyl ^-Gly A21 -des (B30) - human insulin, N ^{&lt; [epsilon] B29} - tridecanoyl -Gly ^{A 1} Gln ^B3 -des (B30) - human insulin, N ^{&lt; [epsilon] B29} - tetradecanoyl ^{-Gly A21 Gln B3 -des (B30)} - human insulin, N ^{&lt; [epsilon] B29} - decanoyl ^{-Gly A21 -Gln B3 -des (B30)} - human insulin , N ^{&lt; [epsilon] B29} - dodecanoyl ^{-Gly A21 -Gln B3 -des (B30)} - human insulin, N ^{&lt; [epsilon] B29} - tridecanoyl ^-Ala A21 -des (B30) - human insulin, N ^{&lt; [epsilon] B29} - tetradecanoyl ^-Ala A21 -des (B30) ^-Human insulin, N [ ^{epsilon] B29} -decanoyl-Ala ^{A21- des} (B30) ^-human insulin, N [ ^{epsilon] B29} -dodecanoyl-Ala ^{A21- des} (B30) ^-human insulin, N [ ^{epsilon] B29} -tridecanoyl-Ala ^{A2 1-} Gln ^B3- des (B30) ^-human insulin, N [ ^{epsilon] B29} -tetradecanoyl-Ala ^A21 Gln ^B3- des (B30) ^-human insulin, N [ ^{epsilon] B29} -decanoyl-Ala ^A21 Gln ^{B3- des} (B30) ^-human insulin ^NεB29 -dodecanoyl-Ala ^A21 Gln ^{B3- des} (B30) ^-human insulin, ^NεB29 -tridecanoyl-Gln ^{B3- des} (B30) ^-human insulin, ^NεB29 -tetradecanoyl-Gln ^B3 - ^des (B30)- human insulin, N ^{&lt; [epsilon] B29} - decanoyl ^-Gln B3 -des (B30) - including one of the human insulin mutant and / or chemically modified - human insulin, N ^{&lt; [epsilon] B29} - dodecanoyl ^-Gln B3 -des (B30).

In certain embodiments, the insulin molecule of the present disclosure, the following insulin molecule: N ^{&lt; [epsilon] B29} - tridecanoyl ^{-Gly A21} - human insulin, N ^{&lt; [epsilon] B29} - tetradecanoyl ^{-Gly A21} - human insulin, N ^{&lt; [epsilon] B29} - decanoyl - Gly ^A21 - human insulin, N ^{&lt; [epsilon] B29} - dodecanoyl ^{-Gly A21} - human insulin, N ^{&lt; [epsilon] B29} - tridecanoyl ^{-Ala A21} - human insulin, N ^{&lt; [epsilon] B29} - tetradecanoyl ^{-Ala A21} - human insulin, N ^{&lt; [epsilon] B29} - decanoyl ^{-Ala A21} - human including one of the human insulin mutant and / or chemically modified - insulin, N ^{&lt; [epsilon] B29} - dodecanoyl ^{-Ala A21.}

In certain embodiments, the insulin molecule of the present disclosure, the following insulin molecule: N ^{&lt; [epsilon] B29} - tridecanoyl ^-Gly A21 ^{Gln B3} - human insulin, N ^{&lt; [epsilon] B29} - tetradecanoyl ^-Gly A21 ^{Gln B3} - human insulin, N ^{&lt; [epsilon] B29} - decanoyl ^-Gly A21 ^{Gln B3} - human insulin, N ^{&lt; [epsilon] B29} - dodecanoyl ^-Gly A21 ^{Gln B3} - human insulin, N ^{&lt; [epsilon] B29} - tridecanoyl ^-Ala A21 ^{Gln B3} - human insulin, N ^{&lt; [epsilon] B29} - tetradecanoyl ^-Ala A21 ^{Gln B3} - One ^{mutation of} human insulin, N [ ^{epsilon] B29} -decanoyl-Ala ^A21 Gln ^B3 -human insulin, N [ ^{epsilon] B29} -dodecanoyl-Ala ^A21 Gln ^B3 -human insulin and / or Includes chemical modification.

In certain embodiments, the insulin molecule of the present disclosure, the following insulin molecule: N ^{&lt; [epsilon] B29} - tridecanoyl -Gln ^B3 - human insulin, N ^{&lt; [epsilon] B29} - tetradecanoyl -Gln ^B3 - human insulin, N ^{&lt; [epsilon] B29} - decanoyl - It includes one mutation and / or chemical modification of Gln ^B3 -human insulin, ^NεB29 -dodecanoyl-Gln ^B3 -human insulin.

In some specific embodiments, an insulin molecule of the present disclosure comprises the following insulin molecules: N ^εB29 -tridecanoyl-Glu ^{B30 -human} insulin, N ^{εB29 -tetradecanoyl} -Glu ^{B30 -human} insulin, N ^{εB29 -decanoyl-} Glu ^B30 - containing one of the human insulin mutant and / or chemically modified - human insulin, N ^{&lt; [epsilon] B29} - dodecanoyl ^{-Glu B30.}

In certain embodiments, the insulin molecule of the present disclosure, the following insulin molecule: N ^{&lt; [epsilon] B29} - tridecanoyl ^-Gly A21 ^{Glu B30} - human insulin, N ^{&lt; [epsilon] B29} - tetradecanoyl ^-Gly A21 ^{Glu B30} - human insulin, N ^{&lt; [epsilon] B29} - containing one of the human insulin mutant and / or chemically modified - decanoyl ^-Gly A21 ^{Glu B30} - human insulin, N ^{&lt; [epsilon] B29} - dodecanoyl ^-Gly A21 ^{Glu B30.} In certain embodiments, the insulin molecule of the present disclosure, the following insulin molecule: N ^{&lt; [epsilon] B29} - tridecanoyl ^-Gly A21 ^Gln B3 ^{Glu B30} - human insulin, N ^{&lt; [epsilon] B29} - tetradecanoyl ^-Gly A21 ^Gln B3 ^{Glu B30} - human insulin, N ^{&lt; [epsilon] B29} - decanoyl ^-Gly A21 ^Gln B3 ^{Glu B30} - human insulin, N ^{&lt; [epsilon] B29} - dodecanoyl ^-Gly A21 ^Gln B3 ^{Glu B30} - human insulin, N ^{&lt; [epsilon] B29} - tridecanoyl ^-Ala A21 ^{Glu B30} - human insulin, N ^{&lt; [epsilon] B29} - tetra Decanoyl-Ala ^A21 Glu ^B30 -human insulin, ^NεB29 -decanoyl-Ala ^A21 Glu ^B30 -human insulin, ^NεB29 -dodecanoyl-Ala ^A21 Glu ^B30 - human insulin, N ^{&lt; [epsilon] B29} - tridecanoyl ^-Ala A21 ^Gln B3 ^{Glu B30} - human insulin, N ^{&lt; [epsilon] B29} - tetradecanoyl ^-Ala A21 ^Gln B3 ^{Glu B30} - human insulin, N ^{&lt; [epsilon] B29} - decanoyl ^-Ala A21 ^Gln B3 ^{Glu B30} - It contains a mutation and / or chemical modification of one of human insulin, ^NεB29 -dodecanoyl-Ala ^A21 Gln ^B3 Glu ^{B30 -human} insulin.

In some specific embodiments, an insulin molecule of the present disclosure comprises the following insulin molecules: N ^{εB29 -tridecanoyl-} Gln ^B3 Glu ^{B30 -human} insulin, N ^{εB29 -tetradecanoyl} -Gln ^B3 Glu ^{B30 -human} insulin, N ^{It includes} one mutation and / or chemical modification of ^εB29 -decanoyl-Gln ^B3 Glu ^{B30 -human} insulin, N ^{εB29 -dodecanoyl-} Gln ^B3 Glu ^{B30 -human} insulin. In some specific embodiments, an insulin molecule of the present disclosure comprises the following insulin molecules: ^NεB29 -formyl-human insulin, ^NαB1 -formyl-human insulin, ^NαA1 -formyl-human insulin, ^NεB29 -formyl- ^NαB1 -formyl-human insulin, ^NεB29 -formyl- ^NαA1 -formyl-human insulin, ^NαA1 -formyl- ^NαB1 -formyl-human insulin, ^NεB29 -formyl- ^NαA1 -formyl- ^NαB1 -formyl-human Includes mutations and / or chemical modifications of one of the insulins.

In some specific embodiments, an insulin molecule of the present disclosure has the following insulin molecules: N [ ^{epsilon] B29} -acetyl-human insulin, N [ ^{alpha] B1} -acetyl-human insulin, N [ ^{alpha] A1} -acetyl-human insulin, N [ ^{epsilon] B29} -acetyl- ^NαB1 -acetyl-human insulin, ^NεB29 -acetyl- ^NαA1- acetyl-human insulin, ^NαA1 -acetyl- ^NαB1 -acetyl-human insulin, ^NεB29 -acetyl- ^NαA1- acetyl- ^NαB1- acetyl-human It includes a mutation and / or chemical modification of one of the insulins.

In some specific embodiments, an insulin molecule of the present disclosure has the following insulin molecules: ^NεB29 -propionyl-human insulin, ^NαB1 -propionyl-human insulin, ^NαA1 -propionyl-human insulin, ^NεB29 -acetyl-. ^NαB1 -propionyl-human insulin, ^NεB29 -propionyl- ^NαA1 -propionyl-human insulin, ^NαA1 -propionyl- ^NαB1 -propionyl-human insulin, ^NεB29 -propionyl- ^NαA1 -propionyl- ^NαB1 -propionyl-human Includes mutations and / or chemical modifications of one of the insulins.

In some specific embodiments, an insulin molecule of the present disclosure has the following insulin molecules: N [ ^{epsilon] B29} -butyryl-human insulin, N [ ^{alpha] B1} -butyryl-human insulin, N [ ^{alpha] A1} -butyryl-human insulin, N [ ^{epsilon] B29-butyryl-} N [ ^{alpha] B1} -butyryl-human insulin, N [ ^{epsilon] B29} -butyryl-N [ ^{alpha] A1} -butyryl-human insulin, N [ ^{alpha] A1} -butyryl-N [ ^{alpha] B1} -butyryl-human insulin, N [ ^{epsilon] B29} -butyryl-N [ ^{alpha] A1} -butyryl-N [ ^{alpha] B1-} butyryl-human It includes a mutation and / or chemical modification of one of the insulins.

In some specific embodiments, an insulin molecule of the present disclosure has the following insulin molecules: ^NεB29 -pentanoyl-human insulin, ^NαB1 -pentanoyl-human insulin, ^NαA1 -pentanoyl-human insulin, ^NεB29 -pentanoyl-. ^NαB1 -pentanoyl-human insulin, ^NεB29 -pentanoyl- ^NαA1 -pentanoyl-human insulin, ^NαA1 -pentanoyl- ^NαB1 -pentanoyl-human insulin, ^NεB29 -pentanoyl- ^NαA1 -pentanoyl- ^NαB1 -pentanoyl-human Includes mutations and / or chemical modifications of one of the insulins.

In some specific embodiments, an insulin molecule of the present disclosure has the following insulin molecules: ^NεB29 -hexanoyl-human insulin, ^NαB1 -hexanoyl-human insulin, ^NαA1 -hexanoyl-human insulin, ^NεB29 -hexanoyl-. ^NαB1 -hexanoyl-human insulin, ^NεB29 -hexanoyl- ^NαA1 -hexanoyl-human insulin, ^NαA1 -hexanoyl- ^NαB1 -hexanoyl-human insulin, ^NεB29 -hexanoyl- ^NαA1 -hexanoyl- ^NαB1 -hexanoyl-human Includes mutations and / or chemical modifications of one of the insulins.

In some specific embodiments, an insulin molecule of the present disclosure comprises the following insulin molecules: ^{NεB29-heptanoyl-} human insulin, ^{NαB1-heptanoyl-} human insulin, ^{NαA1-heptanoyl-} human insulin, ^{NεB29-heptanoyl-} N ^Arufabi1 - heptanoyl - human insulin, N ^{&lt; [epsilon] B29} - heptanoyl -N ^Arufaei1 - heptanoyl - human insulin, N ^αA1 - heptanoyl -N ^Arufabi1 - heptanoyl - human insulin, N ^{&lt; [epsilon] B29} - heptanoyl -N ^Arufaei1 - heptanoyl -N ^Arufabi1 - heptanoyl - human Includes mutations and / or chemical modifications of one of the insulins.

In some specific embodiments, an insulin molecule of the present disclosure comprises the following insulin molecules: ^NαB1 -octanoyl-human insulin, ^NαA1 -octanoyl-human insulin, ^NεB29 -octanoyl- ^NαB1 -octanoyl-human insulin, One ^{mutation of NεB29} -octanoyl- ^NαA1 -octanoyl-human insulin, ^NαA1 -octanoyl- ^NαB1 -octanoyl-human insulin, ^NεB29 -octanoyl- ^NαA1 -octanoyl- ^NαB1 -octanoyl-human insulin and And / or chemical modification.

In some specific embodiments, an insulin molecule of the present disclosure has the following insulin molecules: ^NεB29 -nonanoyl-human insulin, ^NαB1 -nonanoyl-human insulin, ^NαA1 -nonanoyl-human insulin, ^NεB29 -nonanoyl-. ^NαB1 -Nanoyl-human insulin, ^NεB29 -Nanoyl- ^NαA1 -Nanoyl-human insulin, ^NαA1 -Nanoyl- ^NαB1 -Nanoyl-human insulin, ^NεB29 -Nanoyl- ^NαA1 -Nanoyl- ^NαB1 -Nanoyl-human Includes mutations and / or chemical modifications of one of the insulins.

In some specific embodiments, an insulin molecule of the present disclosure has the following insulin molecules: ^NεB29 -decanoyl-human insulin, ^NαB1 -decanoyl-human insulin, ^NαA1 -decanoyl-human insulin, ^NεB29 -decanoyl-. ^NαB1 -decanoyl-human insulin, ^NεB29 -decanoyl- ^NαA1 -decanoyl-human insulin, ^NαA1 -decanoyl- ^NαB1 -decanoyl-human insulin, ^NεB29 -decanoyl- ^NαA1- decanoyl- ^NαB1 -decanoyl-human Includes mutations and / or chemical modifications of one of the insulins.

In certain embodiments, the insulin molecule of the present disclosure, the following insulin ^molecule: N εB28 - formyl ^-Lys B28 ^{Pro B29} - human ^insulin, N αB1 - formyl ^-Lys B28 ^{Pro B29} - human ^insulin, N αA1 - Formyl-Lys ^B28 Pro ^{B29 -human} insulin, ^NεB28 -formyl- ^NαB1 -formyl-Lys ^B28 Pro ^B29 -human insulin, ^NεB28 -formyl- ^NαA1 -formyl-Lys ^B28 Pro ^B29 -human insulin, ^NαA1 -formyl -N ^Arufabi1 - formyl ^-Lys B28 ^{Pro B29} - human ^insulin, N εB28 - formyl -N ^Arufaei1 - formyl -N ^Arufabi1 - formyl ^-Lys B28 ^{Pro B29} - human insulin, N ^{&lt; [epsilon] B29} - acetyl - ys ^B28 Pro ^B29 - human insulin, N ^αB1 - acetyl ^-Lys B28 ^{Pro B29} - human ^insulin, N αA1 - acetyl ^-Lys B28 ^{Pro B29} - human ^insulin, N εB28 - acetyl ^-N αB1 - acetyl ^-Lys B28 ^{Pro B29} - human Includes mutations and / or chemical modifications of one of the insulins.

In certain embodiments, the insulin molecule of the present disclosure, the following insulin ^molecule: N εB28 - acetyl ^-N αA1 - acetyl ^-Lys B28 ^{Pro B29} - human insulin, N ^αA1 - acetyl ^-N αB1 - acetyl -Lys ^B28 Pro ^B29 - containing one of the human insulin mutant and / or chemically modified - human ^insulin, N εB28 - acetyl -N ^Arufaei1 - acetyl -N ^Arufabi1 - acetyl ^-Lys B28 ^{Pro B29.}

In certain embodiments, the insulin molecule of the present disclosure, the following insulin ^molecule: N εB28 - propionyl ^-Lys B28 ^{Pro B29} - human ^insulin, N αB1 - propionyl ^-Lys B28 ^{Pro B29} - human ^insulin, N αA1 - Propionyl-Lys ^B28 Pro ^{B29 -human} insulin, N [ ^{epsilon] B28-} propionyl-N [ ^{alpha] B1} -propionyl-Lys ^B28 Pro ^B29 -human insulin, N [ ^{epsilon] B28-} propionyl-N [ ^{alpha] A1} -propionyl-Lys ^B28 Pro ^B29 -human insulin, N [ ^{alpha] A1 proil -N} αB1 - propionyl ^-Lys B28 ^{Pro B29} - human ^insulin, N εB28 - propionyl -N ^Arufaei1- propionyl ^-N αB1 - propionyl ^-Lys B28 Pro ^{29 -} including the one of the human insulin mutant and / or chemically modified.

In certain embodiments, the insulin molecule of the present disclosure, the following insulin ^molecule: N εB28 - butyryl ^-Lys B28 ^{Pro B29} - human ^insulin, N αB1 - butyryl ^-Lys B28 ^{Pro B29} - human ^insulin, N αA1 - butyryl ^-Lys B28 ^{Pro B29} - human ^insulin, N εB28 - butyryl ^-N αB1 - butyryl ^-Lys B28 ^{Pro B29} - human ^insulin, N εB28 - butyryl ^-N αA1 - butyryl ^-Lys B28 ^{Pro B29} - human insulin, N ^αA1 - butyryl ^-N αB1 - butyryl ^-Lys B28 ^{Pro B29} - human ^insulin, N εB28 - butyryl ^-N αA1 - butyryl ^-N αB1 - butyryl ^-Lys B28 ^{Pro B29} - one of the human insulin mutant and / Or including chemical modification.

In certain embodiments, the insulin molecule of the present disclosure, the following insulin ^molecule: N εB28 - pentanoyl ^-Lys B28 ^{Pro B29} - human ^insulin, N αB1 - pentanoyl ^-Lys B28 ^{Pro B29} - human ^insulin, N αA1 - Pentanoyl-Lys ^B28 Pro ^{B29 -human} insulin, N [ ^{epsilon] B28-} pentanoyl-N [ ^{alpha] B1} -pentanoyl-Lys ^B28 Pro ^B29 -human insulin, N [ ^{epsilon] B28-} pentanoyl-N [ ^{alpha] A1} -pentanoyl-Lys ^B28 Pro ^B29 -human insulin, N [ ^{alpha] A1} -pentanoyl ^-N αB1 - pentanoyl ^-Lys B28 ^{Pro B29} - human ^insulin, N εB28 - pentanoyl ^-N αA1 - pentanoyl ^-N αB1 - pentanoyl ^-Lys B28 Pro ^{29 -} including the one of the human insulin mutant and / or chemically modified.

In certain embodiments, the insulin molecule of the present disclosure, the following insulin ^molecule: N εB28 - hexanoyl ^-Lys B28 ^{Pro B29} - human ^insulin, N αB1 - hexanoyl ^-Lys B28 ^{Pro B29} - human ^insulin, N αA1 - hexanoyl ^-Lys B28 ^{Pro B29} - human ^insulin, N εB28 - hexanoyl ^-N αB1 - hexanoyl ^-Lys B28 ^{Pro B29} - human ^insulin, N εB28 - hexanoyl ^-N αA1 - hexanoyl ^-Lys B28 ^{Pro B29} - human insulin, N ^αA1 - hexanoyl ^-N αB1 - hexanoyl ^-Lys B28 ^{Pro B29} - human ^insulin, N εB28 - hexanoyl ^-N αA1 - hexanoyl ^-N αB1 - hexanoyl ^-Lys B28 Pro ^{29 -} including the one of the human insulin mutant and / or chemically modified.

In certain embodiments, the insulin molecule of the present disclosure, the following insulin ^molecule: N εB28 - heptanoyl ^-Lys B28 ^{Pro B29} - human ^insulin, N αB1 - heptanoyl ^-Lys B28 ^{Pro B29} - human ^insulin, N αA1 - Heptanoyl-Lys ^B28 Pro ^B29 -human insulin, N [ ^{epsilon] B28- heptanoyl-} N [ ^{alpha] B1-heptanoyl-} Lys ^B28 Pro ^B29 -human insulin, N [ ^{epsilon] B28- heptanoyl-} N [ ^{alpha] A1-heptanoyl-} Lys ^B28 Pro ^B29 -human insulin, N [ ^{alpha] A1 heptohe -N} αB1 - heptanoyl ^-Lys B28 ^{Pro B29} - human ^insulin, N εB28 - heptanoyl ^-N αA1 - heptanoyl ^-N αB1 - heptanoyl ^-Lys B28 Pro ^{29 -} including the one of the human insulin mutant and / or chemically modified.

In certain embodiments, the insulin molecule of the present disclosure, the following insulin ^molecule: N εB28 - octanoyl ^-Lys B28 ^{Pro B29} - human ^insulin, N αB1 - octanoyl ^-Lys B28 ^{Pro B29} - human ^insulin, N αA1 - octanoyl ^-Lys B28 ^{Pro B29} - human ^insulin, N εB28 - octanoyl ^-N αB1 - octanoyl ^-Lys B28 ^{Pro B29} - human ^insulin, N εB28 - octanoyl ^-N αA1 - octanoyl ^-Lys B28 ^{Pro B29} - human insulin, N ^αA1 - octanoyl ^-N αB1 - octanoyl ^-Lys B28 ^{Pro B29} - human ^insulin, N εB28 - octanoyl ^-N αA1 - octanoyl ^-N αB1 - octanoyl ^-Lys B28 Pro ^{29 -} including the one of the human insulin mutant and / or chemically modified.

In certain embodiments, the insulin molecule of the present disclosure, the following insulin ^molecule: N εB28 - nonanoyl ^-Lys B28 ^{Pro B29} - human ^insulin, N αB1 - nonanoyl ^-Lys B28 ^{Pro B29} - human ^insulin, N αA1 - Nonanoyl-Lys ^B28 Pro ^{B29 -human} insulin, N [ ^{epsilon] B28-} nonanoyl-N [ ^{alpha] B1} -nonanoyl-Lys ^B28 Pro ^B29 -human insulin, N [ ^{epsilon] B28-} nonanoyl-N [ ^{alpha] A1} -nonanoyl-Lys ^B28 Pro ^B29 -human insulin, N [ ^{alpha] A1} -nonanoyl ^-N αB1 - nonanoyl ^-Lys B28 ^{Pro B29} - human ^insulin, N εB28 - nonanoyl -N ^Arufaei1- nonanoyl ^-N αB1 - nonanoyl ^-Lys B28 ^{Pro B29} - human insulin Chino containing one mutation and / or chemically modified.

In certain embodiments, the insulin molecule of the present disclosure, the following insulin ^molecule: N εB28 - decanoyl ^-Lys B28 ^{Pro B29} - human ^insulin, N αB1 - decanoyl ^-Lys B28 ^{Pro B29} - human ^insulin, N αA1 - decanoyl ^-Lys B28 ^{Pro B29} - human ^insulin, N εB28 - decanoyl ^-N αB1 - decanoyl ^-Lys B28 ^{Pro B29} - human ^insulin, N εB28 - decanoyl ^-N αA1 - decanoyl ^-Lys B28 ^{Pro B29} - human insulin, N ^αA1 - decanoyl ^-N αB1 - decanoyl ^-Lys B28 ^{Pro B29} - human ^insulin, N εB28 - decanoyl ^-N αA1 - decanoyl ^-N αB1 - decanoyl ^-Lys B28 ^{Pro B29} - human insulin Including one of these mutations and / or chemical modifications.

In certain embodiments, the insulin molecule of the present disclosure, the following insulin molecule: N ^{&lt; [epsilon] B29} - pentanoyl ^-Gly A21 ^Arg B31 ^{Arg B32} - human ^insulin, N αB1 - hexanoyl ^-Gly A21 ^Arg B31 ^{Arg B32} - human insulin , N ^αA1 - heptanoyl ^-Gly A21 ^Arg B31 ^{Arg B32} - human insulin, N ^{&lt; [epsilon] B29} - octanoyl ^-N αB1 - octanoyl ^-Gly A21 ^Arg B31 ^{Arg B32} - human insulin, N ^{&lt; [epsilon] B29} - propionyl ^-N αA1 - propionyl ^-Gly A21 ^{Arg B31} Arg ^B32 -human insulin, N [ ^{alpha] A1} -acetyl-N [ ^{alpha] B1} -acetyl- ^{Gly A21} Arg ^B31 Arg ^B32 -human insulin, N [ ^{epsilon] B29} -formyl-N ^{[alpha] A1} -formyl-N [ ^{alpha] B1} -formyl- ^{Gly A21} Arg ^B31 Arg ^{B32 -human} insulin, N [ ^{epsilon] B29} -formyl- ^des (B26) ^-human insulin, N [ ^{alpha] B1} -acetyl-Asp ^B28 -human insulin, N [ ^{epsilon] B29} -propionyl-N [ ^{alpha] A1} - propionyl -N ^Arufabi1 - propionyl ^-Asp B1 ^Asp B3 AspB ²¹ - human insulin, N ^{&lt; [epsilon] B29} - pentanoyl ^{-Gly A21} - human insulin, N ^αB1 - hexanoyl ^{-Gly A21} - human ^insulin, N αA1 - heptanoyl ^{-Gly A21} - human insulin , N ^{&lt; [epsilon] B29} - octanoyl ^-N αB1 - octanoyl ^{-Gly A21} - human insulin, N ^{&lt; [epsilon] B29} - propionyl ^-N αA1 - propionyl ^{-Gly A21} - Hitoinsu Emissions, N ^αA1 - acetyl ^-N αB1 - acetyl ^{-Gly A21} - human insulin, N ^{&lt; [epsilon] B29} - formyl ^-N αA1 - formyl ^-N αB1 - formyl ^{-Gly A21} - human insulin, N ^{&lt; [epsilon] B29} - butyryl -des (B30) - human Insulin, N [ ^{alpha] B1} -butyryl- ^des (B30) ^-human insulin, N [ ^{alpha] A1} -butyryl- ^des (B30) ^-human insulin, N [ ^{epsilon] B29} -butyryl-N [ ^{alpha] B1} -butyryl- ^des (B30) ^-human insulin, N [ ^{epsilon] B29} -butyryl -N [ ^{alpha] A1} -butyryl- ^des (B30) ^-human insulin, N [ ^{alpha] A1} -butyryl-N [ ^{alpha] B1} -butyryl- ^des (B30) ^-human insulin, N [ ^{epsilon] B29} -butyryl-N [ ^{alpha] A1} -butyryl-N [ ^{alpha] B1} -butyryl- ^des (B30). )-Of human insulin Including one of these mutations and / or chemical modifications.

  The present disclosure also encompasses modified forms of non-human insulin (eg, porcine insulin, bovine insulin, rabbit insulin, sheep insulin, etc.) comprising any one of the aforementioned mutations and / or chemical modifications.

  These and other modified insulin molecules are described in US Pat. Nos. 6,906,028; 6,551,992; 6,465,426; 6,444,641; 335,316; 6,268,335; 6,051,551; 6,034,054; 5,952,297; 5,922,675; No. 5,747,642; No. 5,693,609; No. 5,650,486; No. 5,547,929; No. 5,504,188; No. 5,474 No. 5,461,031; and U.S. Pat. No. 4,421,685; and U.S. Pat. No. 7,387,996; No. 6,869,930; No. 6,011,007; No. 5,866,538; and It is described in detail in Nos. No. 5,750,497, the entire disclosure of which is incorporated herein by reference.

  In various embodiments, an insulin molecule of the present disclosure has three wild type disulfide bonds (ie, one between position 7 of A chain and position 7 of B chain, between position 20 of A chain and position 19 of B chain). And the third between the 6th and 11th positions of the A chain).

  A method for conjugating drugs containing insulin molecules is described below. In some specific embodiments, the insulin molecule is conjugated to the conjugate framework via the A1 amino acid residue. In some specific embodiments, the A1 amino acid residue is glycine. However, it will be appreciated that the present disclosure is not limited to N-terminal conjugation and that in some specific embodiments, insulin molecules may be conjugated via non-terminal A-chain amino acid residues. In particular, the present disclosure encompasses conjugation via the ε-amine group of a lysine residue (introduced by wild type or site-directed mutagenesis) present at any position within the A chain. It will be appreciated that different conjugation positions on the A chain can result in different reductions in insulin activity. In some specific embodiments, the insulin molecule is conjugated to the conjugate framework via the B1 amino acid residue. In some specific embodiments, the B1 amino acid residue is phenylalanine. However, it will be appreciated that the present disclosure is not limited to N-terminal conjugation, and in some specific embodiments, insulin molecules may be conjugated via non-terminal B-chain amino acid residues. In particular, the present disclosure encompasses conjugation via ε-amine groups of lysine residues (introduced by wild type or site-directed mutagenesis) present at any position within the B chain. For example, in some specific embodiments, the insulin molecule can be conjugated via a B29 lysine residue. In the case of insulin gluridine, conjugation to the framework of the conjugate via the B3 lysine residue can be used. It will be appreciated that different conjugation positions on the B chain can result in different decreases in insulin activity.

In some specific embodiments, the ligand is conjugated to more conjugation points of one drug, such as an insulin molecule. For example, an insulin molecule can be conjugated to both the N-terminus of A1 and B29 lysine. In some embodiments, amide conjugation occurs in carbonate buffer and is conjugated at the B29 and Al positions but not at the B1 position. In other embodiments, the insulin molecule can be conjugated to the N-terminus of A1, the N-terminus of B1, and B29 lysine. In still other embodiments, protecting groups are used such that conjugation occurs at the B1 and B29 positions or the B1 and Al positions. It will be appreciated that any combination of conjugation points on the insulin molecule can be used. In some embodiments, at least one of the conjugation points is a mutated lysine residue, such as Lys ^A3 .

  In various embodiments, the conjugate can include an insulin sensitivity improving agent (ie, a drug that enhances the action of insulin). Drugs that enhance the effects of insulin include biguanides (eg, metformin) and glitazones. The first glitazone drug was troglitazone, which was found to have severe side effects. Second generation glitazones include pioglitazone and rosiglitazone, which are better tolerated, but rosiglitazone has been associated with adverse cardiovascular events in some trials.

  In various embodiments, the conjugate may include an insulin secretagogue (ie, a drug that stimulates insulin secretion by pancreatic β cells). For example, in various embodiments, the conjugate can include a sulfonylurea. Sulfonylurea stimulates the secretion of insulin by pancreatic β cells by sensitizing the cells to the action of glucose. Furthermore, sulfonylureas can inhibit glucagon secretion and sensitize target tissues to the action of insulin. First generation sulfonylureas include tolbutamide, chlorpropamide and carbutamide. Second generation sulfonylureas active at lower doses include glipizide, glibenclamide, gliclazide, glibornuride and glimepiride. In various embodiments, the conjugate can comprise meglitinide. Suitable meglitinides include nateglinide, mitiglinide and repaglinide. Their hypoglycemic action is faster but shorter than that of sulfonylureas. Other insulin secretagogues include glucagon-like peptide 1 (GLP-1) and GLP-1 analogs (i.e. having GLP-1-like physiological activity, with 1-10 amino acid substitutions with GLP-1) , Peptides differing by addition or deletion and / or chemical modification). GLP-1 reduces food intake by suppressing gastric emptying through central action to increase satiety and by suppressing glucagon release. GLP-1 decreases plasma glucose levels by increasing islet cell proliferation and increasing insulin production after feeding. GLP-1 can be chemically modified, for example, by conjugation of lipids as in the case of liraglutide to increase its in vivo half-life. Other insulin secretagogues include exendin-4 and exendin-4 analogs (ie, exendin-4-like physiological activity, with exendin-4 being a substitution, addition or deletion of 1-10 amino acids. And / or peptides that differ by chemical modification). Exendin-4 is found in poison in the American monster monster (Gila Monster) and exhibits GLP-1-like physiological activity. It has a much longer half-life than GLP-1, and unlike GLP-1, the N-terminal 8 amino acid residues can be cleaved without loss of bioactivity. The N-terminal regions of GLP-1 and exendin-4 are nearly identical, with a significant difference being the second amino acid residue, alanine for GLP-1, and glycine for exendin-4, which results in exendin-4 Confers resistance to in vivo digestion. Exendin-4 also has nine more amino acid residues at the C-terminus when compared to GLP-1. Mann et al. Biochem. Soc. Trans. 35: 713-716, 2007 and Runge et al., Biochemistry 46: 5830-5840, 2007 describe various GLP-1 and exendin-4 analogs that can be used in the conjugates of the present disclosure. The short half-life of GLP-1 is due to enzymatic digestion by dipeptidyl peptidase IV (DPP-IV). In some specific embodiments, the effects of endogenous GLP-1 can be enhanced by administration of a DPP-IV inhibitor (eg, vildagliptin, sitagliptin, saxagliptin, linagliptin or alogliptin).

  In various embodiments, the conjugate is amylin or an amylin analog (ie, a peptide having amylin-like bioactivity and differing from amylin by 1-10 amino acid substitutions, additions or deletions and / or chemical modifications). May be included. Amylin plays an important role in glucose regulation (see, eg, Edelman and Weyer, Diabetes Technol. Ther. 4: 175-189, 2002). Amylin is a neuroendocrine hormone that is co-secreted with insulin by pancreatic beta cells in response to food intake. Insulin serves to regulate the disappearance of glucose from the bloodstream, whereas amylin serves to help regulate the appearance of glucose from the stomach and liver into the bloodstream. Pramlintide acetate (SYMLIN®) is an exemplary amylin analog. Because natural human amylin is amyloidogenic, strategies for designing pramlintide involved replacing certain residues with residues from rat amylin that are not amyloidogenic. In particular, proline residues have been found to be structurally destructive residues, so they were grafted directly from the rat sequence to the human sequence. Glu-10 was also substituted with asparagine.

  In various embodiments, the preconjugate drug is one that includes one or more reactive moieties (eg, moieties such as carboxyl or reactive ester, amine, hydroxyl, aldehyde, sulfhydryl, maleimidyl, alkynyl, azide, etc.). obtain. As discussed below, such reactive moieties may facilitate the conjugation process in some specific embodiments. Specific examples include peptide drugs having an α-terminal amine and / or an ε-amine lysine group. It will be appreciated that any such reactive moiety can be artificially added to a known drug if not already present. For example, in the case of a peptide drug, an appropriate amino acid (eg, lysine) can be added to or substituted within the amino acid sequence. It will also be appreciated that the conjugation process can be controlled by selectively blocking certain reactive moieties prior to conjugation, as discussed in more detail below.

  As discussed above, the present disclosure is not limited to any particular drug / exogenous target molecule combination.

Conjugate frameworks Conjugates can be prepared from frameworks that naturally contain affinity ligands (eg, polysaccharides such as glycogen and dextran naturally contain glucose affinity ligands) and / or affinity It can be prepared by artificially incorporating the ligand into a natural or synthetic framework. It will be appreciated that the conjugates of the present disclosure are not limited to a particular framework structure. For example, conjugates can be prepared using framework structures that include polymeric structures and / or non-polymeric structures. It will also be appreciated that the framework structure of the conjugate can be linear, branched, hyperbranched and / or combinations thereof. The following sections show some exemplary conjugate framework structures.

  In various embodiments, the conjugate can be prepared from a framework structure comprising a polymer structure. For example, polymers with reactive pendant groups (eg, carboxyl or reactive esters, amines, hydroxyls, aldehydes, sulfhydryls, maleimidyl, alkynyls, azides, etc.) can be used. It will be appreciated that different pendant groups may be mixed within a single framework (eg, polymer frameworks are obtained by copolymerizing the appropriate monomers in the desired ratio). As discussed below, such reactive groups can be used to attach affinity ligands and / or drugs to the framework. Different framework copolymers, mixtures and adducts may also be used. Such a combination may be useful for optimizing the mechanical and chemical properties of the material.

  In various embodiments, a framework with a carboxyl (or reactive ester) pendant group (—COOH-containing framework, or CBF) can be used. Such a framework structure may naturally contain a carboxyl group or may be modified to contain a carboxyl group. Exemplary polymer CBFs include, but are not limited to, carboxylated polysaccharides (CPS), such as alginate (Ag), carboxymethylated-D-manno-D-glucan (CMMG, available from Daiichi Pharmaceutical Co.), Examples include carboxymethyl dextran (CMDex), carboxymethyl chitin (CMCh, available from Katakura Chikkalin Co.), N-desulfated N-acetylated heparin (DSH), and hyaluronic acid (HA). DSH and CMDex are described in Sugahara et al., Biol. Pharm. Bull, 24, 535-543 (2001). In general, hydroxylated frameworks can be carboxylated by reaction with chloroacetic acid under basic conditions. For polymer framework structures, the degree of carboxyl substitution on the monomers can vary between 1 and 100 mol%. Naturally occurring carboxylated polymers include, but are not limited to, carboxylated poly (amino acids) (CPAA) such as poly-L-glutamate and poly-L-aspartate. The carboxylate content includes carboxylated amino acids (eg, amino acids having a carboxyl group in addition to carboxyl groups that are part of the polymer backbone) and non-carboxylated amino acids (eg, only carboxyl groups are part of the polymer backbone). Can be varied between 1 to 100% mol COOH / mol AA residues.

In various embodiments, a framework with an amine pendant group (—NH ₂ containing framework, or NBF) can be used. Such a framework structure may exist naturally, or may be chemically modified to include a primary amine. The latter include, but are not limited to, polymer frameworks such as amine pendant polysaccharides (NPS) (such as deacetylated chitosan (Ch) (Sigma Aldrich, Milwaukee, Wis.)) And diethylaminoethyl ether dextran (DEAEDex), MW 500,000 g / mol (Polysciences, Warrington, Pa.). For such a polymer framework structure, the degree of amine substitution on the monomer can vary between 1 and 100 mol%. Other suitable NBFs include, but are not limited to, polynucleotides in which one or more of the purine bases at the 2 ′ position is derivatized with an amine group. Naturally occurring aminated polymers include, but are not limited to, poly (amino acids) such as poly-L-lysine (PLL) and its enantiomers. The amine content can be determined by comparing aminated amino acids (eg, amino acids that have amines in addition to amine groups that will eventually become part of the polymer backbone) and non-aminated amino acids (eg, where only amines are ultimately polymer backbones). Can be varied between 1 to 100% mol NH ₂ / mol amino acid residues.

  In various embodiments, polymers with hydroxyl pendant groups (—OH containing frameworks, or OBFs) can be used. Such a framework structure may be naturally hydroxylated or chemically modified to include a hydroxyl group. In addition to dextran, naturally occurring polymer OBFs include, but are not limited to, yeast mannan (Mn), pullulan (Pl), amylose (Am), amylopectin (AmP), glycogen (Gl), cellulose (Cl), hyaluro Examples include polysaccharides such as Nate (Hy), Chondroitin (ChD), and Dextrin (Dx), all of which are commercially available from Sigma Aldrich. Poly (amino acids) such as poly (serine), poly (threonine), poly (tyrosine), and poly (4-hydroxyproline) can also be used as hydroxylated polymers. The hydroxyl content of poly (amino acids) can be varied between 1-100% mol-OH / mol amino acid residues by copolymerizing hydroxylated amino acids with non-hydroxylated amino acids. Of course, the carboxyl (or reactive ester), amino, and hydroxyl pendant groups may be mixed within a single polymer by copolymerizing the appropriate amino acids in the desired ratio.

  In various embodiments, a framework structure with a sulfhydryl pendant group (-SH containing framework structure, or SBF) may be used. The SBF may be naturally sulfhydrylated or chemically modified using standard organic chemistry techniques to include a sulfhydryl group. In other embodiments, framework structures with pendant groups such as aldehyde, maleimidyl, alkynyl, azide, etc. can be used.

  In addition to the aforementioned types of framework structures, some exemplary polymers that may be used include poly (lactic acid) (PLA), poly (glycolic acid) (PGA), PLA-PGA copolymer (PLGA), poly ( Anhydrides), poly (hydroxy acids), poly (orthoesters), poly (propyl fumarate), poly (caprolactone), polyamides, polyacetals, biodegradable polycyanoacrylates and biodegradable polyurethanes.

In various embodiments, conjugates of the following general formula (I) may be used.

Various embodiments of conjugates of formula (IV) are described in detail in Example 9, but in general,
R ^x is hydrogen or optionally substituted C _1-6 alkyl;
Z ¹ is an optionally substituted divalent C _1-10 hydrocarbon chain, wherein ¹ , 2, 3, 4, or 5 methylene units of Z ¹ are optionally independently ^{Te, -S -, - O -,} - NR a -, - (C = NR a) -, - (C = O) -, - (S = O) -, - S (= O) 2 -, - (CR ^b = CR ^b )-,-(N = N)-, substituted with one or more groups selected from an optionally substituted arylene moiety or an optionally substituted heteroarylene moiety Wherein R ^a is hydrogen, optionally substituted aliphatic, optionally substituted heteroaliphatic, optionally substituted aryl, optionally substituted heteroaryl. or a suitable amino protecting group, R ^b is an aliphatic hydrogen, it is optionally substituted Heteroaliphatic is optionally substituted, heteroaryl which is substituted aryl or optionally substituted with optionally;
Each X ¹ present is independently —OR ^C or —N (R ^d ) ₂ , wherein R ^c is hydrogen, optionally substituted aliphatic, optionally substituted. A heteroaliphatic, optionally substituted aryl, optionally substituted heteroaryl, a suitable hydroxyl protecting group, cationic group, or affinity ligand, wherein each R ^d is independently hydrogen An optionally substituted aliphatic, an optionally substituted heteroaliphatic, an optionally substituted aryl, an optionally substituted heteroaryl, a suitable amino protecting group, or affinity A ligand, but at least two of the X ¹ present shall comprise an affinity ligand;
Y ¹ is hydrogen, halogen, optionally substituted aliphatic, optionally substituted heteroaliphatic, optionally substituted aryl, optionally substituted heteroaryl, —OR ^e or —SR ^e , wherein R ^e is hydrogen, optionally substituted aliphatic, optionally substituted heteroaliphatic, optionally substituted aryl, or optionally A heteroaryl substituted with
r is an integer from 5 to 25 (inclusive);
W ¹ is a drug;

It should be understood that corresponds to a covalent single or double bond.

In various embodiments, the following general formula (II) conjugates may be used:

(Where:
[(AT)] each represents a possible branch in the conjugate;
(AT) each represents a possible repeat in the branch of the conjugate;
-A- are each independently substituted with a covalent bond, carbon atom, heteroatom, or optionally selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, heteroaryl and heterocyclic. Is a group;
Each T is independently a C _1-30 hydrocarbon chain that is covalently bonded or optionally substituted with a divalent linear or branched saturated or unsaturated, wherein One or more methylene units of T are optionally independently and independently selected from —O—, —S—, —N (R) —, —C (O) —, —C (O) O—, —OC ( O) -, - N (R ) C (O) -, - C (O) N (R) -, - S (O) -, - S (O) 2 -, - N (R) SO 2 -, -SO ₂ N (R) -, heterocyclic group, have been replaced by an aryl group or a heteroaryl group;
Each R is independently hydrogen, a suitable protecting group, or an acyl, arylalkyl, aliphatic, aryl, heteroaryl or heteroaliphatic moiety;
-B is an ^-T-L B -X;
Each X is independently an affinity ligand;
Each ^LB is independently a covalent bond or a group derived from a covalent conjugation of T and X;
-D is -T-L ^D -W;
Each W is independently a drug;
L ^D is each independently a covalent bond or a group derived from a covalent conjugation of T and W;
k is an integer from 2 to 11 (inclusive) and specifies that there are at least two k-branches in the conjugate;
q is an integer from 1 to 4 (inclusive);
k + q is an integer from 3 to 12 (inclusive);
each p is independently an integer from 1 to 5 (inclusive);
each n is independently an integer from 0 to 5 (inclusive);
each m is independently an integer from 1 to 5 (inclusive);
each v is independently an integer from 0 to 5 (inclusive), but at each k-branch, at least one n present is ≧ 1, and at least one v present is ≧ 1 ).

It will be understood that the general formula (II) (and other formulas herein) does not specify every single hydrogen. For example, the center of -A- is a C ₆ aryl group, when a k + q <6, the empty position on the C ₆ aryl ring (s) will recognize that the hydrogen is contained.

  In general, -A- each represents a possible branching node, and the number of branches in each node is determined by the value of the central -A- k and the non-central -A- n value. Be recognized. Since k ≧ 2, the conjugate always contains at least two k-branches. Those skilled in the art will envision both branched and hyperbranched (eg, dendrimer-like) embodiments of such conjugates in this disclosure, as the presence of each n can be an integer from 0 to 5. Will be recognized. At each k-branch, any conjugate requires that at least one n present is ≧ 1 and at least one v present is ≧ 1, B (ie affinity ligand) ) In the presence of at least two separate k-branches.

In some specific embodiments, each -A- present in the p-bracket portion is replaced with a number of n-bracket portions corresponding to a value of n ≧ 1. For example, if k = 2 and p = 2 at both k-branches, the conjugate is of formula (IIa):

Can be.

In other embodiments, only -A- present at the end in the p-bracket portion is replaced with a number of n-bracket portions corresponding to a value of n ≧ 1. For example, if k = 2 and p = 2 in both k-branches (and the first p-bracket part in both k-branches is n = 0), the conjugate can be of formula (IIb ):

Can be.

In some specific embodiments, each -A- present in the m-bracket portion is replaced with a number of B portions corresponding to a value of v ≧ 1. For example, when k = 2, each p = 1 present, and each m = 2 present, the conjugate has formula (IIc):

Can be.

In other embodiments, only -A- present at the end in the m-bracket part is replaced with a number of B parts corresponding to a value of v ≧ 1. For example, if K = 2, each p = 1 present, and each m = 2 present (and v = 0 for the first m-bracket part in each n-branch), the conjugate is Formula (IId):

Can be.

As a further example, when q = 1 and n = 1 in both k-branches of the previous formula, the conjugate is of formula (IIe):

Can be.

Alternatively, if q = 1 and n = 2 in both k-branches of the previous formula, the conjugate has formula (IIf):

Can be.

  In various embodiments, the present disclosure also provides conjugates comprising affinity ligands and / or drugs non-covalently bound to the conjugate framework.

For example, in some embodiments, the disclosure provides that: -A-, T, D, k, q, k + q, p, n, m, and v are each as described above and herein. Define;
-B is -T-LRP ^B -X;
Each X is independently an affinity ligand;
LRP ^B is each independently a ligand-receptor pair that forms a non-double bond between T and X, and the dissociation constant in human serum is less than 1 pmol / L.
Conjugates of any of the foregoing formulas are provided.

In still other embodiments, the present disclosure provides that -A-, T, B, k, q, k + q, p, n, m and v are each defined as described above and herein. ;
-D is -T-LRP ^D -W;
Each W is independently a drug or a detectable label;
Each LRP ^D is independently a ligand-receptor pair that forms a non-double bond between T and W, and the dissociation constant in human serum is less than 1 pmol / L.
Conjugates of any of the foregoing formulas are provided.

In other embodiments, according to the present disclosure, -A-, T, k, q, k + q, p, n, m and v are each defined as described above and herein;
-B is -T-LRP ^B -X;
Each X present is independently an affinity ligand;
Each LRP ^B present is independently a ligand-receptor pair that forms a non-double bond between T and X, and the dissociation constant in human serum is less than 1 pmol / L;
-D is -T-LRP ^D -W;
Each W present is independently a drug;
Each LRP ^D present is independently a ligand-receptor pair that forms a non-double bond between T and W, and the dissociation constant in human serum is less than 1 pmol / L.
Conjugates of any of the foregoing formulas are provided.

（式中、−Ａ−、Ｂ、Ｔ、Ｄ、ｖ、ｍ、ｎおよびｐは本明細書において定義および記載のとおりであり、ｋは、１〜１１（両端を含む）の整数であり、ｊは、２〜４（両端を含む）の整数である）を有するものであり得る。式（ＩＩＩ）のコンジュゲートは、薬物に対するリガンドのコンジュゲーション部位を多数有するものであり得る。ｑが１である場合、式中、ｊが２、３または４である式（ＩＩＩ）のコンジュゲートに対して、当業者には、上記のもの（式（ＩＩａ）〜（ＩＩｆ））に対して示される類似した亜属が想定され得ることは認識されよう。 In various embodiments, the conjugates of the present disclosure have the general formula (III):

Wherein -A-, B, T, D, v, m, n and p are as defined and described herein, k is an integer from 1 to 11 (inclusive), j may be an integer from 2 to 4 (inclusive). The conjugate of formula (III) may have a large number of ligand conjugation sites for the drug. When q is 1, for those conjugates of formula (III) where j is 2, 3 or 4, the skilled person will It will be appreciated that similar subgenera shown may be envisioned.

For illustrative purposes and for avoidance of confusion, -A-D-A- (ie when j is 2) present in the conjugate of formula (III) is replaced with -A-T-L ^{D- W-L} D ^-T-A- (where the drug is covalently bonded to the framework structure of the conjugate) or ^{-A-T-LRP D -W-} LRP D -T-A- ( drug conjugate framework It should be understood that this can be expressed as (when non-covalently bound to the structure).

Exemplary Group Description -A- (Section)
In some specific embodiments, each occurrence of -A- is optionally substituted independently selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, heteroaryl and heterocyclic. It is a group that has been. In some embodiments, each -A-
Are the same. In some embodiments, the central -A- is different from all other -A-. In some specific embodiments, all -A- are the same except for the central -A-. In some embodiments, -A- is an optionally substituted aryl or heteroaryl group. In some embodiments, -A- is 6-membered aryl. In some specific embodiments, -A- is phenyl. In some specific embodiments, -A- is a heteroatom selected from N, O, or S. In some embodiments, -A- is a nitrogen atom. In some embodiments, -A- is an oxygen atom. In some embodiments, -A- is a sulfur atom. In some embodiments, -A- is a carbon atom.

T (spacer)
In some specific embodiments, each T present is independently a divalent linear or branched saturated or unsaturated, optionally substituted C _1-20 hydrocarbon chain. And one or more methylene units of T are optionally and independently, —O—, —S—, —N (R) —, —C (O) —, —C (O) O—, — OC (O) -, - N (R) C (O) -, - C (O) N (R) -, - S (O) -, - S (O) 2 -, - N (R) SO 2 _{-, - SO 2 N (R} ) -, it is replaced by a heterocyclic group, an aryl group or a heteroaryl group. In some specific embodiments, 1, 2, 3, 4 or 5 methylene units of T are optionally replaced independently. In some specific embodiments, T is C _1-10 , C _1-8 , C _1-6 , C _1-4 , C _2-12 , C _4-12 , C _6-12 , C _{8- 12} or C _10-12 hydrocarbon chains, wherein one or more methylene units of T are optionally independently -0-, -S-, -N (R)- , -C (O)-, -C (O) O-, -OC (O)-, -N (R) C (O)-, -C (O) N (R)-, -S (O) _{-, - S (O) 2} -, - N (R) SO 2 -, - SO 2 N (R) -, is replaced by a heterocyclic group, an aryl group or a heteroaryl group. In some embodiments, one or more methylene units of T are replaced with a heterocyclic group. In some embodiments, one or more methylene units of T are replaced with a triazole moiety. In some specific embodiments, one or more methylene units of T are replaced with -C (O)-. In some specific embodiments, one or more methylene units of T are replaced with —C (O) N (R) —. In some specific embodiments, one or more methylene units of T are replaced with -0-.

In some embodiments, T is

It is.

In some embodiments, T is

It is.

In some embodiments, T is

It is.

In some embodiments, T is

It is.

In some embodiments, T is

It is.

In some embodiments, T is

It is.

  In some specific embodiments, each T is the same.

（式中、−Ａ−、Ｂ、Ｄ、ｖ、ｍ、ｎ、ｐ、ｋおよびｊは、それぞれ、式（ＩＩ）または（ＩＩＩ）で定義および記載のとおりである）のものである。 In some specific embodiments, each T (outside of the B and D groups) is a double bond, and the conjugate has the general formula (V) or (VI):

Wherein -A-, B, D, v, m, n, p, k and j are as defined and described in formula (II) or (III), respectively.

In some specific embodiments of general formula (V) or (VI), each -A- is a double bond except for the central -A-, each v is 1, and the conjugate is of the formula (VII) or (VIII):

Wherein -A-, B, D, q, k and j are as defined and described in formula (II) or (III), respectively.

  In certain such embodiments of formula (VII), k = 2 and q = 1.

  In other embodiments, k = 3 and q = 1.

  In other embodiments, k = 2 and q = 2.

  In certain such embodiments of formula (VIII), k = 1 and j = 2.

  In other embodiments, k = 2 and j = 2.

  In other embodiments, k = 3 and j = 2.

  In other embodiments, k = 1 and j = 3.

  In other embodiments, k = 2 and j = 3.

  In other embodiments, k = 3 and j = 3.

In some embodiments, the present disclosure provides for general formula (VIIa):

Wherein B and D are as defined and described herein, are provided.

For example, in some embodiments, the present disclosure provides the formula:

Wherein W and X are as defined and described herein, are provided.

In some embodiments, the present disclosure provides for general formula (VIIb):

Wherein B and D are as defined and described herein, are provided.

For example, in some embodiments, the present disclosure provides the formula:

Wherein W and X are as defined and described herein, are provided.

In some embodiments, the present disclosure provides for general formula (VIIc):

Wherein B and D are as defined and described herein, are provided.

For example, in some embodiments, the present disclosure provides the formula:

Wherein W and X are as defined and described herein, are provided.

For conjugates of formula (VIII) where j is 2, 3 or 4, those skilled in the art will recognize subgenus similar to those of formula (VIIa), (VIIb) and (VIIc) and one of them. It will be appreciated that can be assumed. For example, when j is 2, in some specific embodiments, according to this disclosure, the formula:

Wherein B and D are as defined and described herein, are provided.

In some specific embodiments, the disclosure provides the formula:

Wherein W, X and j are as defined and described herein, are provided.

B (Ligand)
In various embodiments, -B is -T-L B ^-X, wherein, X is a ligand; L ^B is covalent conjugation to either a covalent bond, or X and T Is a group derived from Exemplary ligands are described above.

D (drug)
In various embodiments, -D is -T-L ^D -W, wherein W is a drug and L ^D is a covalent bond or derived from a covalent conjugation of W and T. It is a group to do. Exemplary drugs were as described above.

L ^B and L ^D (covalent conjugation)
One skilled in the art will recognize that various conjugation chemistries can be used for covalent conjugation of X and T and / or W and T (generally “components”). Such techniques are widely known in the art, and exemplary techniques are discussed below. The component may be directly attached (ie, without intervening chemical groups) and a cup (eg, a cup that provides some degree of physical isolation between the conjugate element and the remainder of the conjugate framework). It may be indirectly linked via a ring agent or a covalent bond chain). It is understood that the component can be covalently bound to the conjugate framework by any number of chemical bonds (eg, but not limited to, amide, amine, ester, ether, thioether, isourea, imine, etc. bonds). Like. In some specific embodiments, L ^B and / or L ^D (generally “L” as interpreted in this section) is a double bond. In some embodiments, L is an optionally substituted carbonyl-reactive, thiol-reactive, amine-reactive, or hydroxyl-reactive moiety of T, such as X or W carboxyl, thiol, amine, or hydroxyl. An optionally substituted moiety derived from conjugating with a group. In some embodiments, L is an optionally substituted carboxyl-reactive, thiol-reactive, amine-reactive, or hydroxyl-reactive moiety of X or W, and the carboxyl, thiol, amine or hydroxyl of T An optionally substituted moiety derived from conjugating with a group. In some embodiments, L is

It is. In some embodiments, L is a succinimide moiety.

In various embodiments, the component can be covalently attached to the conjugate framework using a “click chemistry” reaction known in the art. Such reactions include, for example, cycloaddition reactions, nucleophilic ring-opening reactions, and additions to carbon-carbon multiple bonds (eg, Kolb and Sharpless, Drug Discovery Today 8: 1128-1137, 2003 and the like). And the cited references and Dondoni, Chem. Asian J. 2: 700-708, 2007, and references cited therein). As discussed above, in various embodiments, the component can be attached to the conjugate framework via a natural or chemically attached pendant group. In general, the first and second members of a pair of reactive groups (eg, a carboxyl group and an amine group that react to produce an amide bond) can be present in either the component or the framework ( That is, it will be appreciated that the relative positions of the two members are not important as long as they react to give a conjugate. Exemplary combinations are discussed in more detail below. In various embodiments, the carboxyl (or reactive ester) containing component is converted to an —OH containing framework (OBF) using the procedure outlined in Kim et al., Biomaterials 24: 4843-4851 (2003). Can be conjugated. Briefly, OBF is dissolved in DMSO with carboxyl-containing components and reacted in a dry atmosphere with N ′, N′-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) as catalysts. Carboxyl-containing components can be conjugated to —NH ₂ -containing frameworks (NBF) using a carbodiimide (EDAC) coupling procedure. Using this procedure, the carboxyl-containing component is functionalized by reaction with EDAC in a pH 5 buffer and then added to NBF. In any such case (and in any of the following cases), the resulting product may be obtained by any number of means available to those skilled in the art, including, but not limited to, size exclusion chromatography, reverse phase chromatography. And purified by silica gel chromatography, ion exchange chromatography, ultrafiltration, and selective precipitation.

  In various embodiments, the amine-containing component can be coupled to a —COOH-containing framework (CBF). CBF uses activated ester moieties (see, eg, Hermanson in Bioconjugate Techniques, 2nd edition, Academic Press, 2008 and references cited therein). Briefly, CBF having a terminal activated carboxylic acid ester (eg, —NHS, —SSC, —NPC, etc.) is dissolved in an anhydrous organic solvent (eg, DMSO or DMF). The desired equivalent number of amine-containing components is then added and mixed for several hours at room temperature. Amine-containing components are also described in Baudys et al., Bioconj. Chem. 9: 176-183, 1998 can be conjugated to CBF to obtain a stable amide bond. This reaction can be performed by adding tributylamine (TBA) and isobutyl chloroformate to a solution of CBF and amine-containing components in dimethyl sulfoxide (DMSO) under anhydrous conditions. Alternatively, the amine-containing component is OBF, including but not limited to cyanogen bromide (CNBr), N-cyanotriethylammonium tetrafluoroborate (CTEA), 1-cyano-4- (dimethylamino) -pyridinium tetrafluoroborate ( Coupling may be via cyanation using reagents such as fluorborate (CDAP) and p-nitrophenyl cyanate (pNPC). The CNBr reaction can be performed in an aqueous solution at a weakly basic pH. The CDAP reaction is carried out in a mixture of DMSO and water at a weakly basic pH using triethylamine (TEA) as a catalyst. In some specific embodiments, the amine-containing component can be conjugated to NBF, for example by quenching with glycine after glutaraldehyde coupling in an aqueous buffer solution containing pyridine. In some specific embodiments, an amine-containing component can be conjugated to an aldehyde-containing framework by reduction after use of a Schiff base coupling procedure (eg, Hermanson, Bioconjugate Technologies, 2nd edition, Academic. Press, 2008 and references cited therein, and Mei et al., Pharm. Res. 16: 1680-1686, 1999 and references cited therein). Briefly, frameworks with terminal activated aldehydes (eg, acetaldehyde, propionaldehyde, butyraldehyde, etc.), aqueous buffer with neutral or lower pH (to suppress undesired aldehyde hydrolysis) Dissolve in liquid. The desired equivalent number of amine-containing components are then added and mixed at room temperature before excess suitable reducing agent (eg, sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride pyridine borane, triethylamine borane). Etc.).

  In various embodiments, the hydroxyl-containing component can be conjugated to OBF according to the divinyl sulfone (DVS) procedure. Using this procedure, OBF is added to pH 11.4 bicarbonate buffer, activated with DVS, followed by the addition of hydroxyl-containing components, followed by the addition of glycine to neutralize and quench the reaction. . A hydroxyl-containing component may also be coupled to OBF using the activated ester moiety described above that provides an ester linkage.

In various embodiments, a sulfhydryl-containing component can be coupled to a maleimide-containing framework (MBF) using a relatively mild procedure that results in a thioether bond (eg, Hermanson, Bioconjugate Technologies, 2nd edition, Academic Press). , 2008 and references cited therein). Since maleimide groups are much less hydrolyzed than activated esters, the reaction may be carried out under aqueous conditions. Briefly, MBF is dissolved in an aqueous buffer solution at pH 6.5-7.5, and then the desired number of equivalent sulfhydryl-containing components are dissolved. After mixing for several hours at room temperature, the thioether coupling conjugate can be purified. Sulfhydryl-containing components are also described in Toma et al. Am. Chem. Soc. 121: 5919-5929, 1999 may be conjugated to NBF. This reaction involves suspending NBF in anhydrous dimethylformamide (DMF) followed by addition of 2,6-lutidine and acid anhydride followed by purification of the reactive intermediate. The sulfhydryl-containing component is then added to the intermediate in DMF (including triethylamine). In various embodiments, when an azide-containing component is coupled to an alkyne-containing framework (ABF) using a copper (I) -catalyzed modern version of the Huisgen-type azide-alkyne cycloaddition, -Substituted 1,2,3-triazoles can be obtained (see, for example, Dondoni, Chem. Asian J. 2: 700-708, 2007 and references cited therein, and Dedola et al., Org. Biomol. Chem. 5: 1006-1017, 2007). This reaction is commonly referred to as the “click” reaction and can be performed, for example, in undiluted THF using N, N-diisopropylethylamine and Cu (PPhS) ₃ Br as the catalyst system (eg, Wu et al. Chem. Commun. 5775-5777, 2005). This reaction can also be carried out in a 3: 1 (THF: water) mixture using sodium ascorbate and CuSO ₄ .5H ₂ O as a catalyst system (see, eg, Wu et al., Supra). In either case, the azide-containing component is added to the ABF in the desired number of equivalents and then mixed at room temperature for 12 to 48 hours. Alternatively, the alkyne-containing component may be conjugated to an azide-containing framework structure using exactly the same conditions as described above.

  Some specific such components may be naturally occurring having more than one same chemically reactive moiety. In some examples, the chemical reaction type and conditions can be selected such that the component reacts selectively at only one of the sites. For example, when insulin is conjugated via a reactive amine, in some specific embodiments, the N-terminal α-Phe-B1 retains insulin bioactivity while maintaining N-terminal α-Gly- Preferred binding sites over A1 and ε-Lys-B29 (see, eg, Mei et al., Pharm. Res. 16: 1680-1686, 1999 and references cited therein, and Tsai et al., J. Pharm. Sci. 86: 1264-1268, 1997). In an exemplary reaction of insulin with hexadecenal (an aldehyde-terminated molecule), researchers have determined that the two components are 1.5M pH 6.8 containing 54% isopropanol in the presence of sodium cyanoborohydride. Mixing overnight in a 1: 6 (insulin: aldehyde mol / mol) ratio in aqueous sodium salicylate results in over 80% conversion to a single substituted Phe-B1 secondary amine conjugate product. (Mei et al., Pharm. Res. 16: 1680-1686, 1999). Their study showed that the choice of solvent, pH, and insulin: aldehyde ratio all affected the selectivity and yield of the reaction. However, in most cases, it is difficult to obtain selectivity by selecting chemical reaction conditions. Thus, in some specific embodiments, the component (eg, insulin) at all sites other than the one site desired for the reaction is selectively protected, followed by reacting and purifying the substance. It may be convenient to perform a deprotection step later. For example, there are many examples of selective protection of insulin amine groups available in the literature, which can be deprotected under acidic (BOC), weakly acidic (citraconic anhydride), and basic (MSC) conditions. (See, for example, Tsai et al., J. Pharm. Sci. 86: 1264-1268, 1997; Dixon et al., Biochem. J. 109: 312-314, 1968; Physiol.Chem.360: 1721,1979). In one example, the amines of Gly-A1 and Lys-B29 can be selectively protected with a tert-butoxycarbonyl (BOC) group, which is then in a solution of 90% trifluoroacetic acid (TFA) / 10% anisole. Removed after conjugation by incubation for 1 hour at 4C. In one embodiment, a dry powder of insulin is dissolved in anhydrous DMSO and then excess triethylamine is dissolved. To this solution is slowly added approximately 2 equivalents of di-tert-butyl bicarbonate solution (in THF) and the solution is mixed for 30-60 minutes. After the reaction, the crude solution is poured into excess acetone and then diluted HCl is added dropwise to precipitate the reacted insulin. The precipitate is centrifuged, washed with acetone and completely dried under vacuum. The desired di-BOC protected product is separated from unreacted insulin, undesired di-BOC isomers, and mono-BOC and tri-BOC byproducts using preparative reverse phase HPLC or ion exchange chromatography. (See, for example, Tsai et al., J. Pharm. Sci. 86: 1264-1268, 1997). For reverse phase HPLC, load the crude product in 70% water / 30% acetonitrile (containing 0.1% TFA) onto a C8 column and elute with an increasing gradient of acetonitrile. Collect the desired di-BOC peak, spin concentrate to remove acetonitrile, and lyophilize to obtain the pure product.

LRP ^B and LRP ^D (non-covalent conjugation)
One skilled in the art will recognize that a variety of conjugation chemistries can be used for non-covalent conjugation of X and T and / or W and T (generally “components”). Such techniques are widely known in the art, and exemplary techniques are discussed below. In some specific embodiments, the non-covalent dissociation constant (K _d ) in human serum is less than 1 pmol / L. For example, the component can be non-covalently bound to the conjugate framework by a non-covalent ligand-receptor pair well known in the art (eg, but not limited to a biotin-avidin pair). In such embodiments, one member of the ligand-receptor pair is covalently bound to the component and the other member of the pair is covalently bound to the conjugate framework. When the component and conjugate framework are combined, a strong non-covalent interaction between the ligand and its receptor results in the component being non-covalently bound to the conjugate framework. Typical ligand / receptor pairs include protein / cofactor and enzyme / substrate pairs. In addition to commonly used biotin / avidin pairs, such pairs include, but are not limited to, biotin / streptavidin, digoxigenin / anti-digoxigenin, FK506 / FK506 binding protein (FKBP), rapamycin / FKBP, cyclophilin / cyclosporine, and A glutathione / glutathione transferase pair may be mentioned. Other suitable ligand / receptor pairs such as, but not limited to, glutathione-S-transferase (GST), c-myc, FLAG®, and Kessler pp. 105-152, Advances in Mutagenesis ”, edited by Kessler, Springer-Verlag, 1990;“ Affinity Chromatography: Methods and Protocols (Methods in Molecular Bio; Those skilled in the art will recognize monoclonal antibodies in pairs with epitope tags such as those described in “Techniques”, Academic Press, 1992.

k and q
In the conjugate of general formula (II), k is an integer from 2 to 11 (inclusive), defining that there are at least two k-branches in the conjugate. In some specific embodiments, k = 2 or 3. q is an integer of 1 to 4 (including both ends), and defines the number of D groups bonded to the central -A- group. In some specific embodiments, q = 1. In some embodiments, q = 2. k + q is an integer of 3 to 6 (including both ends). In some specific embodiments, k + q = 3 or 4.

  In the conjugate of the general formula (III), when j is 2, 3 or 4, k is an integer of 1 to 11 (including both ends). In some specific embodiments, k is 1, 2 or 3. q is an integer of 1 to 4 (including both ends), and defines the number of D groups bonded to the central -A- group. In some specific embodiments, q = 1. In some embodiments, q = 2. k + q is an integer of 3 to 6 (including both ends). In some specific embodiments, k + q = 3 or 4.

p and m
Each p present is independently an integer of 1 to 5 (inclusive). In some specific embodiments, each p present is the same. In some specific embodiments, p = 1, 2, or 3. In some specific embodiments, p = 1.

  Each existing m is independently an integer of 1 to 5 (including both ends). In some specific embodiments, each m present is the same. In some specific embodiments, m = 1, 2, or 3. In some specific embodiments, m = 1.

n and v
Each existing n is independently an integer of 0 to 5 (including both ends), but at each k-branch, at least one of the existing n is ≧ 1. A branch within a given k-branch is referred to herein as an n-branch.

  In some specific embodiments, each -A- present in the p-bracket portion is substituted with a number of n-bracket portions corresponding to a value of n ≧ 1 (eg, Formula (IIa) above). checking). In some such embodiments, each n present in the conjugate is the same. In some of such embodiments, n = 1 or 2.

  In other embodiments, only -A- present at the terminus within the p-bracket part is substituted with a number of n-bracket parts corresponding to a value of n ≧ 1 (eg, Formula (IIb) above) checking). In some specific embodiments, each k-branch includes only one occurrence of n ≧ 1 (ie, all other n = 0 present). In some such embodiments, each n present in the conjugate is the same. In some of such embodiments, n = 1 or 2.

  Each existing v is independently an integer of 0 to 5 (inclusive), but at each k-branch, at least one of the existing v is ≧ 1.

  In some specific embodiments, each of -A- present in the m-bracket moiety is substituted with a number of B moieties corresponding to a value of v ≧ 1 (see, eg, Formula (IIc) above) thing). In some such embodiments, each v present in the conjugate is the same. In some of such embodiments, v = 1 or 2.

  In other embodiments, only -A- present at the end in the m-bracket part is substituted with a number of B parts corresponding to a value of v ≧ 1 (see, eg, formula (IId) above) thing). In some specific embodiments, each k-branch includes only one occurrence of v ≧ 1 (ie, all other v = 0 present). In some such embodiments, each v present in the conjugate is the same. In some of such embodiments, v = 1 or 2. In some specific embodiments, each n-branch has at least one occurrence of v ≧ 1 included. In some specific embodiments, each n-branch includes only one occurrence of v ≧ 1 (ie, all other v = 0 present). In some such embodiments, each v present in the conjugate is the same. In some of such embodiments, v = 1 or 2.

j
J in formula (II) is an integer from 1 to 4 (inclusive) and defines the number of conjugations to the D group. In some specific embodiments, j = 1. In some specific embodiments, j = 2. In some embodiments, j = 3. In other embodiments, j = 4.

Drug loading In general, the number of drugs loaded into a conjugate depends on the molecular weight of the drug, and the molecular weight and / or chemical activation of the conjugate framework (ie, when a pendant group is added to the framework). Can be controlled by adjusting the level of. In various embodiments, the drug loading level can range from 5 to 99% w / w drug to conjugate (ie, including drug). In various embodiments, load levels within a narrower range of 50-99% (eg, a range of 80-99%) may be used.

Others In various embodiments, a biodegradable framework may be used. In various embodiments, for example, if biodegradability is not relevant to the application, and / or if the resulting framework or conjugate is sufficiently well excreted and biodegradability is not required, A degradable framework structure may be used. In various embodiments, the framework of the conjugate (or spacer (eg when present between the drug and the framework)) is sensitive to enzymatic digestion. In various embodiments, the enzyme is present at the site of administration. One skilled in the art will recognize that there are several enzymes in the patient that can cleave the conjugate framework. Such, but not limited to, include saccharidases, peptidases, and nucleases. Exemplary saccharidases include, but are not limited to, maltase, sucrase, amylase, glucosidase, glucoamylase, and dextranase. Exemplary peptidases include, but are not limited to, dipeptidyl peptidase-IV, prolyl endopeptidase, prolidase, leucine aminopeptidase, and glycylglycine dipeptidase. Exemplary nucleases include, but are not limited to, deoxyribonuclease I, ribonuclease A, ribonuclease T1, and nuclease Sl.

One skilled in the art will also recognize that, depending on the choice of enzyme, there are several conjugate framework structures that are sensitive to enzymatic cleavage. For example, if saccharidase degradation is desired, a framework comprising a polysaccharide can be used (eg, but not limited to a conjugate comprising a polysaccharide comprising a repeating chain of 1,4-linked α-D-glucose residues. , Degraded by α-amylase). Non-limiting examples of suitable polysaccharides include any number of sources such as, but not limited to, sweet corn, oysters, liver (human, cow, rabbit, rat, horse), muscle (rabbit leg, rabbit abdomen, Examples include glycogen and partially digested glycogen from fish, rats), rabbit hair, slipper limpet, baker's yeast, and fungi. Other polysaccharide polymers and spacers that may be used include carboxylated polysaccharides, —NH ₂ pendant polysaccharides, hydroxylated polysaccharides, alginate, collagen-glycosaminoglycans, collagen, mannan, amylose, amylopectin, cellulose, hyaluronate , Chondroitin, dextrin, chitosan and the like. If peptidase cleavage is desired, a polypeptide comprising an amino acid sequence recognized by the cleavage enzyme can be used (eg, but not limited to a conjugate comprising a [-glycine-proline-] sequence is degraded by prolidase. ). In some specific embodiments, a copolymer of an aminated amino acid and a non-aminated amino acid, a copolymer of a hydroxylated amino acid and a non-hydroxylated amino acid, a copolymer of a carboxylated amino acid and a non-carboxylated amino acid, a copolymer of the above, or of the above Adducts can be used. Where nuclease degradation is desired, polynucleotides can be used (eg, but not limited to conjugates comprising polynucleotides comprising oligomers of consecutive adenosine residues are degraded by ribonuclease A).

In various embodiments, the pharmacokinetic and / or pharmacodynamic behavior of a conjugate (ie, a conjugated drug and / or a drug released from the conjugate by chemical or enzymatic degradation) is determined by a corresponding non-conjugate. It can be substantially the same as the gated drug (eg, both administered subcutaneously). For example, from a pharmacokinetic (PK) perspective, the serum concentration curve may be substantially the same as when an equivalent amount of unconjugated drug is administered. Additionally or alternatively, serum T _max , serum C _max , mean serum residence time (MRT), mean serum absorption time (MAT) and / or serum half-life are substantially the same as when non-conjugated drugs are administered. Can be the same. From a pharmacodynamic (PD) standpoint, the conjugate can act on body material in substantially the same way as an unconjugated drug. For example, in the case of insulin conjugates, the conjugates can have a substantially similar effect on blood glucose levels as unconjugated insulin. In this case, a substantially similar pharmacodynamic behavior is that the time to reach the minimum blood glucose concentration (T- _bottom ), the blood glucose level is below a certain percentage of the initial value (eg, 70% of the initial value). Or it can be observed by comparing, for example, the duration maintained at a state of T _{70% BGL} ). It will be appreciated that such PK and PD properties can be measured according to any of a variety of published pharmacokinetic and pharmacodynamic methods (eg, Baudys et al., Bioconjugate Chem for methods suitable for subcutaneous delivery). 9: 176-183, 1998).

In one embodiment, for a conjugate (ie, an isolated form without a crosslinker), a pharmacokinetic (PK) parameter (maximum serum drug concentration is within 40% of the value measured with the unconjugated drug. Time to reach (T _max ), mean drug residence time (MRT), serum half-life, mean drug absorption time (MAT), etc. are obtained. In various embodiments, the conjugate provides a PK parameter that is within 35%, 30%, 25%, 20%, 15%, or even 10% of that obtained with an unconjugated drug. In some embodiments, the conjugate provides a PK parameter that is within 20% of that obtained with an unconjugated drug. For example, in embodiments relating to insulin conjugates for subcutaneous delivery, the conjugate may have an insulin T _{maximum of} 15-30 minutes, an average insulin residence time (MRT) of less than 50 minutes, or an average insulin absorption time of less than 40 minutes ( MAT) can be obtained, all within 20% of the value measured in the human recombinant insulin treatment group. In some specific embodiments, the conjugate can provide an insulin T _{maximum of} 20-25 minutes, an average insulin residence time (MRT) of less than 45 minutes, and an average insulin absorption time (MAT) of less than 35 minutes. . In some specific embodiments, the conjugate can provide a serum half-life of less than 120 minutes, such as less than 100 minutes.

In one embodiment, in the conjugate of the invention, the time to reach the minimum / maximum blood concentration of the substance (T- _bottom / T _maximum ) or the blood level of the substance is below 70% of the initial value / above 130%. Pharmacodynamic (PD) parameters such as the duration ( _{T70% BL} / _{T130% AL} ) of maintaining state are obtained. For example, in an embodiment relating to an insulin conjugate for subcutaneous delivery, the conjugate can yield a glucose T _{bottom of} 45-60 minutes and a glucose T _{70% BL of} less than 180 minutes, both of which are human recombinant Within 20% of the value measured in the insulin treatment group. In some specific embodiments, the conjugate can yield a glucose T _{bottom of} 50-55 minutes and a glucose T _{70% BL of} less than 160 minutes. In various embodiments, PD parameters that are within 40%, 35%, 30%, 25%, 20%, 15%, or even 10% of that obtained with non-conjugated drugs are obtained in various embodiments. In some embodiments, the conjugated provides a PD parameter that is within 20% of that obtained with an unconjugated drug.

Intermediates for the preparation of conjugates In one aspect, the invention provides reagents for preparing conjugates of the present disclosure. Accordingly, in various embodiments, the general formula (II):
Wherein -A-, T, D, k, q, k + q, p, n, m and v are each defined as described above and herein; B is -TLB ^' Is;
Each LB ^′ present is independently hydrogen, an alkyne-containing moiety, an azide-containing moiety, or an optionally substituted carbonyl-reactive, thiol-reactive, amine-reactive, or hydroxyl-reactive moiety) Are provided.

In other embodiments, the general formula (II):
Wherein -A-, T, B, k, q, k + q, p, n, m and v are each defined as described above and herein;
D is -T-L ^{D '} ;
Each ^{LD '} present is independently hydrogen, an alkyne-containing moiety, an azide-containing moiety, or an optionally substituted carbonyl-, thiol-reactive, amine-reactive, or hydroxyl-reactive moiety) Are provided.

Methods for Preparing Conjugates As described in this example, we have included insulin as an exemplary drug and aminoethylglucose (AEG), aminoethylmannose (AEM), amino as exemplary affinity ligands. Examples of methods for preparing the conjugates were given using ethylbimannose (AEBM) and / or aminoethyltrimannose (AETM). Without limitation, conjugates having two affinity ligands and one drug molecule, with short distances between all framework components, are tris (hydroxymethyl) aminomethane (Tris), Tris-succinimi Dilaminotriacetate (TSAT), tris-succinimidyl-1,3,5-benzenetricarboxylate (TSB), and benzene-1,3,5-tricarboxy- (N-4-butyric acid-NHS-ester) amide ( TSB-C4) can be prepared using a conjugate framework. If additional spacing between framework components is desired, succinimidyl (6-aminocaproyl) aminotriacetate (TSAT-C6), succinimidyl (6-amino (PEO-6)) aminotriacetate (TSAT-PEO- 6), benzene-1,3,5-tricarboxy- (N-6-aminocaproic acid-NHS ester) amide (TSB-C6), and benzene-1,3,5-tricarboxy- (N-10-10) Aminodecanoic acid-NHS ester) amide (TSB-C10) can be used. The chemical nature of the TSAT-C6 spacer arm confers more hydrophobic properties to the conjugate compared to TSAT-PEO-6. For example, for illustrative purposes, in one embodiment, both affinity ligands (eg, AEG, AEM, AEMB, and AETM) and insulin are reacted to the TSAT-C6 framework via terminal activated esters. Insulin-TSAT-C6-AEG-2, insulin-TSAT-C6-AEM-2, insulin-TSAT-C6-AEMB-2, and insulin-TSAT-C6-AETM-2 conjugates can be obtained. Various affinity ligands are pre-synthesized as discussed in this example. Also, the A1 and B29 amino groups of insulin are BOC-protected so that each insulin can only react with the Phe-B1 α-amino group as described in this example. Approximately 1 equivalent of BOC-insulin is added as a 40-50 mg / ml DMSO solution at room temperature to 50 mg / ml TSAT-C6 in DMSO containing excess triethylamine and allowed to react for approximately 1 hour. Next, excess AEG, AEM, AEBM and / or AETM (2-10 equivalents) is added as a 100 mg / ml DMSO solution and allowed to react for another 2 hours. After the reaction, this DMSO solution is ultra-diluted 10-fold in a pH 5 physiological saline buffer, then the pH is adjusted to 8.0, and the solution is passed through a Biogel P2 column to remove low molecular weight reactants and salts. . After the material eluted in the void fraction was concentrated using a 3K ultrafiltration device, it was purified on a preparative scale reverse phase HPLC column (C8, acetonitrile / water mobile phase containing 0.1% TFA). The desired product is purified from unreacted BOC2-insulin. The desired elution peak is collected, pooled, and rotoevaporated to remove acetonitrile and then lyophilized to obtain a dry powder. Finally, this lyophilized powder was dissolved in 90% TFA / 10% anisole at 4C for 1 hour, and then diluted more than 10 times in HEPES buffer (pH 8.2) containing 0.150M NaCl to obtain BOC. Remove the protecting group. After adjusting the pH to 7.0-8.0 using NaOH solution, the material is passed through a Biogel P2 column to remove anisole, BOC, and other contaminating salts. The aqueous solution of deprotected purified conjugate is then concentrated to the desired level and stored at 4C until needed.

  Using this exemplary procedure, replacing the TSAT-C6 framework structure with a different framework structure (described below), the spacing between the components of the framework structure is different for different affinity ligands and drugs, for different conjugation chemistries. It will be appreciated that other conjugates can be made that are different and / or have different valences.

For example, if an additional distance is required between the framework components and / or a conserved charge is required at the conjugation site, an appropriately sized amine-containing diethyl acetal (eg, aminopropionaldehyde diethyl After conjugating acetal (APDA) or aminobutyraldehyde diethyl acetal (ABDA)) to one of the reactive groups on the framework described herein, the remaining reactive groups and subject affinity The reaction with a ligand (eg, AEM, AEBM, or AETM) may be completed. Subsequent revelation of the reactive aldehyde group from the diethyl acetal under acidic conditions followed by reductive amination with insulin can complete the drug conjugation step, and then ABDA-TSAT, ABDA-LCTSSAT Etc. can be used. In another example, tetrakis- (N-succinimidylcarboxypropyl) pentaerythritol (TSPE) is used, and three affinity ligands and one drug molecule can be combined for increased multivalentity. It will also be appreciated by those skilled in the art that hyperbranched (eg, dendrimer-like) conjugates with higher order valencies can be made using any of the above teachings. For example, by Roeckendorf and Lindhorst, Topics in Current Chemistry. 217: 202-238, 2001 provides a comprehensive overview of current approaches for making hyperbranched structures. Furthermore, a ligand already having a predetermined degree of multivalence may be reacted again according to the above procedure to obtain higher order ligand multiplicity. For example, a divalent AEM-2, AEBM-2, or AETM-2 molecule containing a reactive terminal amine conjugates each of the two affinity ligands to an appropriate framework that is also conjugated with a reactive amine. Can be prepared. A trivalent AEM-3, AEBM-3, or AETM-3 molecule containing a reactive terminal amine can be obtained by conjugating each of the three affinity ligands to an appropriate framework that is also conjugated with a reactive amine. Can be prepared. NH 2 _- When a bivalent saccharide is reacted with the same framework structure as described above, drug conjugates with drug molecules per one 4-6 ligands can be obtained. NH 2 _- When a trivalent saccharide is reacted with the same framework structure as described above, drug conjugates with drug molecules per one 6-9 pieces of the ligand can be obtained.

  In all cases, a mixture of different ligands is conjugated to the same drug via the framework structure by adjusting the chemistry, valency, and ligand of the multivalent framework structure and the stoichiometry of the framework structure. Recognize that it can be done. For example, insulin-AEM-1-AEBM-1, insulin-AEBM-1-AETM-1, insulin AEM-2-AETM-2, and insulin AEM-1-AETM-2 are all synthesized according to this mixed ligand method Can be done.

  Finally, in some cases it may be desirable to conjugate the affinity ligand to the framework structure by means different from the drug. For example, a divalent maleimide / active monovalent ester functionalized framework (eg, succinimidyl-3,5-dimaleimidophenylbenzoate (SDMB)) contains two sulfhydryl functionalized affinity ligands and one amine function. Can be used to conjugate conjugated drugs in separate steps. For example, insulin or another amine-containing drug can be conjugated to the activated ester moiety of the framework using the methods described herein. In a separate step, aminoethyl saccharides (AEM, AEBM, AETM) may be converted to terminal sulfhydryl-containing ligands by reaction with 4-iminothiolane. Finally, mixing the framework-di-maleimide-insulin conjugate with excess sulfhydryl-functionalized saccharide can result in the formation of a divalent saccharide-insulin conjugate.

Multivalent Crosslinker In some specific embodiments, conjugates of the present disclosure can be combined with a multivalent crosslinker to form a crosslinked material body. The following section describes exemplary crosslinkers that may be used.

  As discussed in more detail below and illustrated in FIG. 4, cross-linked material body 10 is capable of controllably releasing conjugate 20 in response to an exogenous target molecule. The material body combines the conjugate 20 with a multivalent cross-linking agent 30 that binds non-covalently to the affinity ligand 40 of the conjugate 20, thereby cross-linking the conjugate 20 to form a cross-linked material body 10. It is prepared by. Non-covalent bonds between the multivalent crosslinker 30 and the affinity ligand 40 are competitively dissociated in the presence of excess amounts of exogenous target molecules.

1. Polypeptide Crosslinkers In various embodiments, the multivalent crosslinker can comprise a polypeptide. As discussed in more detail below, suitable multivalent polypeptides are naturally occurring (eg, various lectins), but many monovalent binding proteins, such as monovalent lectins, peptides It can also be constructed by linking aptamers, antibodies, cell membrane receptors and the like. Still other multivalent polypeptides may be constructed by chemically linking such protein binding fragments.

  A variety of monovalent and multivalent ligand binding proteins are commercially available (eg, from Sigma-Aldrich) and include several lectins, folate binding proteins, thyroxine binding globulins, lactoferrin, and the like. DeWolf and Best include ligand binding proteins such as biotin binding protein, lipid binding protein / hydrophobic molecule transporter, bacterial periplasmic binding protein, lectin, serum albumin, immunoglobulin, inactivated enzyme, odorant binding protein, An overview of immunosuppressant binding proteins, as well as phosphate- and sulfate binding proteins has been shown (see De Wolfe and Best, Pharm. Rev. 52: 207-236, 2000 and references cited therein. ). In addition, cell membrane receptors for various hormones have been reported in the art. In some specific embodiments, the monovalent or multivalent binding protein is a complex of an existing ligand binding protein after rational design by computer, as described by Looger et al., Nature 423: 185-190, 2003. It can be synthesized by site-directed mutagenesis. Exemplary protein fragments include truncated MBP (Eda et al., Biosci. Biotechnol. Biochem., 62: 1326-1331, 1998), truncated conglutinin (Eda et al., Biochem. J. 316: 43, 1996), cleavage Type SP-D (Eda et al., Biochem. J. 323: 393, 1997) and E. coli glucose / galactose binding protein (Salins et al., Analytical Biochemistry 294: 19-26, 2001).

a. Lectins In some specific embodiments, monovalent or multivalent lectins can be included in the multivalent crosslinker. As discussed in more detail below, in some specific embodiments it may be advantageous to chemically modify the lectin. Lectins are particularly suitable for use in materials designed to respond to sugars (eg, α-methyl-mannose). Lectins are isolated from a variety of natural sources such as seeds, roots, bark, fungi, bacteria, seaweed, sponges, mollusks, fish eggs, invertebrates and lower vertebrate body fluids, and mammalian cell membranes. (See, eg, The Lectins: Properties, Functions, and Applications in Biology and Medicine, edited by Liener et al., Academic Press, 1986). Several lectins have also been produced recombinantly (see, for example, Streicher and Sharon, Methods Enzymol. 363: 47-77, 2003 and US Patent Publication No. 20060247154). As mentioned above, lectins bind to carbohydrates and polysaccharides with a high degree of specificity. For example, some lectins bind only to mannose or glucose residues, while others recognize only galactose residues. Some lectins require certain residues to be in terminal positions, while others bind to residues within the polysaccharide chain. Some lectins require specific anomeric structures, and others recognize specific glycan sequences. The structure and properties of lectins are extensively described in the literature. For a recent review is, Lectins, Sharon and Lis eds., Kluwer Academic Publishers, 2003; Handbook of Animal Lectins: Properties and Biomedical Applications, Kilpatrick ed., Wiley, 2000; and Handbook of Plant Lectins: Properties and Biomedical Applications, Van Damme et al. Ed., Wiley, 1998. Exemplary lectins include calnexin, calreticulin, CD22, CD33, galectin (galactose binding lectin), myelin related glycoprotein, N-acetylglucosamine receptor, selectin, sialoadhesin, aggrecan, asialoglycoprotein receptor, CD94, collectin (Mannose binding lectin), mannose receptor, versican, abrin, lysine, concanavalin A, phytohemagglutinin and pokeweed mitogen. In various embodiments, human analogs of plant lectins can be used. Such as, but not limited to, human mannan-binding protein (MBP, also referred to as mannan-binding lectin, Sheriff et al., Structural Biology, 1: 789-794 (1994); : 465-473 (2002)), human lung surfactant protein A (SP-A, Allen et al., Infection and Immunity, 67: 4563-4569 (1999)), human lung surfactant protein D (SP-D, Personson et al., The Journal of Biological Chemistry, 265: 5755-5760 (1990)), CL-43 (human serum protein), and Nguruchinin and the like.

b. Peptide Aptamers In some specific embodiments, monovalent peptide aptamers can be included in the multivalent crosslinker. As is well known in the art, a peptide aptamer consists of a variable ligand binding peptide loop fused into a protein backbone (eg, Hoppe-Seyler and Butz, J. Mol. Med. 78: 426-430). , 2000 and Crawford et al., Briefings in Functional Genomics and Proteomics 2: 72-79, 2003). This variable loop typically contains about 10-20 amino acids. A variety of scaffold proteins can be used. In general, the insertion site is selected such that in the absence of the peptide loop, a region in the backbone that would mediate some wild-type function is interrupted by the loop (eg, the variable loop is reducible). Bacterial protein thioredoxin-A inserted in the active site (-Cys-Gly-Pro-Cys-loop in wild type protein)). Peptide aptamers with appropriate affinity for the target molecule can be prepared and selected using any known method. For example, a yeast two-hybrid library, yeast expression library, bacterial expression library and / or retroviral library for expression in mammalian cells can be used.

  In various embodiments, peptide aptamers can be selected by affinity chromatography. According to such an embodiment, peptide aptamers in the library are exposed to the target molecule and those that do not bind to the target are removed. The bound peptide aptamer is then eluted and cloned for subsequent selection rounds. A new library is then generated from one or more of the peptide aptamers (eg, the peptide aptamer having the highest affinity for the target molecule in the first round of selection) and the desired binding affinity and Increase or alter the stringency of the elution conditions so that peptide aptamers with specificity are identified. In various embodiments, the selection process can involve gradually increasing the stringency of the elution conditions to select peptide aptamers with high affinity for the target molecule. In various embodiments, the selection process can involve altering elution conditions to select peptide aptamers with the desired specificity for the target molecule (eg, using different affinity columns). By). In various embodiments, the selection process creates a collection of subordinate libraries (or “pools”) that contain peptide aptamers, each with similar affinity and / or specificity for the target molecule. possible. In various embodiments, the selection process can create a single peptide aptamer sequence (or “monoclonal”). It will be appreciated that any such peptide aptamer sequence can be cloned for subsequent recombinant expression.

c. Production of multivalent crosslinkers Multivalent crosslinkers can be made by linking two or more monovalent binding proteins covalently or non-covalently into a single construct. Typically, two or more proteins (which may have the same or different sequences) can be directly linked to each other (eg, by a coupling agent) or framework It can be indirectly linked through a structure. In various embodiments, 2, 3, 4, 5, 6, 7 or 8 or more proteins can be combined in a single construct. In various embodiments, 2, 3, 4, 5, 6, 7 or 8 or more proteins may have the same sequence. It will be appreciated that in any one such approach, it may be necessary to chemically modify the protein prior to coupling (eg, to include a reactive pendant group). Also, the polyvalent crosslinking agents of the present disclosure are not limited to a particular coupling reaction or framework structure (eg, the polyvalent crosslinking agent is prepared using a framework structure that includes a polymer structure and / or a non-polymer structure). It will be appreciated that it may be. Furthermore, it will be appreciated that the framework structure may be linear, branched, dendritic and / or combinations thereof. Exemplary framework structures and coupling chemistries are described below in the context of the conjugate.

  In various embodiments, monovalent binding proteins are covalently bound to each other or to a framework structure. In such embodiments, the proteins may be directly linked (ie, without intervening chemical groups) or spacers (eg, some physical between the proteins or between the proteins and the framework). It may be indirectly linked via a coupling agent or covalent chain that provides sequestration. As discussed below in the context of the conjugate, the proteins are shared with each other or with the framework by any number of chemical bonds, including but not limited to amide, ester, ether, isourea and imine bonds. It will be understood that they can be combined.

In various embodiments, two or more monovalent binding proteins can be non-covalently bound to each other or to a framework structure. In some specific embodiments, the non-covalent dissociation constant (K _d ) in human serum is less than 1 pmol / L. For example, the proteins can be non-covalently bound to each other or to the framework by non-covalent ligand-receptor pairs well known in the art (eg, but not limited to biotin-avidin pairs). In such embodiments, one member of a ligand receptor pair is covalently bound to one protein and the other member of the pair is covalently bound to the other protein or framework. When the proteins (or the protein and the framework structure) are combined, the proteins are non-covalently bound to each other (or to the framework structure) by a strong non-covalent interaction between the ligand and its receptor. become. Typical ligand / receptor pairs include protein / cofactor and enzyme / substrate pairs. In addition to commonly used biotin / avidin pairs, such pairs include, but are not limited to, biotin / streptavidin, digoxigenin / anti-digoxigenin, FK506 / FK506 binding protein (FKBP), rapamycin / FKBP, cyclophilin / cyclosporine, and A glutathione / glutathione transferase pair may be mentioned. Other suitable ligand / receptor pairs such as, but not limited to, glutathione-S-transferase (GST), c-myc, FLAG®, and Kessler pp. 105-152, Advances in Mutagenesis "Kessler ed., Springer-Verlag, 1990;" Affinity Chromatography: Methods and Protocols (Methods in Molecular Biology) ", Pascal Baillon ed., Humana Press, 2000; and Hermanson et al.," Immobilized Affinity Ligand Techniques Monoclonal antibodies in pairs with epitope tags such as those described in Academic Press, 1992 will be recognized by those skilled in the art.

2. Polynucleotide Crosslinkers In various embodiments, the multivalent crosslinker can comprise a polynucleotide aptamer. A polynucleotide aptamer binds to a target molecule and is multivalent (ie, can bind to more than one target molecule). In general, a monovalent aptamer is first prepared based on its binding properties to the target molecule. As is well known in the art, aptamers to various target molecules can be made by an in vitro selection process. See Ellington and Szostak (1990) Nature 346: 818; Tuerk and Gold (1990) Science 249: 505; and US Pat. No. 5,582,981. Also, US Provisional Patent Application No. 61 / 162,092 (filed on March 20, 2009) and corresponding PCT application (filed on January 27, 2010), each of which is incorporated herein by reference. See also the polynucleotide aptamer described in.

  Typically, this process begins with the synthesis of a library of fixed length, randomly generated polynucleotide sequences flanked by 5 'and 3' ends that serve as primers. In some specific embodiments (eg, when optimizing an aptamer), a collection of polynucleotides starting with a sequence known to bind to the target molecule and exhibiting a limited range of change from the starting sequence A library containing (eg, a random set of single mutations) may be generated. The sequences in the library are then exposed to the target molecule and those that do not bind to the target are removed (eg, by affinity chromatography). The bound sequences are then eluted and amplified in preparation for a subsequent selection round (eg, after cloning, by transcription or by PCR), where the desired binding affinity and / or specificity is selected. Increase or alter the stringency of the elution conditions so that sequences with are identified. Jarosch et al. (2006) Nucleic Acids Res. 34:86 describes a method by which the process can be performed without the constant primer region.

  In various embodiments, the selection process may involve gradually increasing the stringency of the elution conditions to select aptamers with high affinity for the target molecule.

  In various embodiments, the selection process can involve changing the elution conditions to select aptamers with the desired specificity for the target molecule (eg, by using different affinity columns). ).

  In various embodiments, the selection process is to create a collection of sub-libraries (or “pools”) that contain aptamers, each with similar affinity and / or specificity for the target molecule. obtain. In various embodiments, the selection process can be to create a single aptamer sequence (or “monoclonal”). In various embodiments, the aptamer is a DNA system.

  In various embodiments, the aptamer is an RNA system. In various embodiments, the aptamer is a mixed RNA / DNA aptamer. Multivalent aptamers can be made by linking two or more such monovalent aptamers covalently or non-covalently into a single construct. An exemplary method is described in Example 4 below. Typically, two or more aptamers (which may have the same sequence or different sequences) can be directly linked to each other (eg, by a coupling agent) or independent It can be indirectly connected through the framework structure. In various embodiments, 2, 3, 4, 5, 6, 7 or 8 aptamers can be combined in a single construct. In various embodiments, 2, 3, 4, 5, 6, 7 or 8 aptamers can have the same sequence. It will be appreciated that in any one of such approaches, it may be necessary to chemically modify the aptamer prior to coupling (eg, to include a reactive pendant group). In addition, the aptamers of the present disclosure are not limited to a particular coupling reaction or framework structure (eg, the aptamer may be prepared using a framework structure including a polymer structure and / or a non-polymer structure). ) Will also be recognized. Furthermore, it will be appreciated that the framework structure may be linear, branched, dendritic and / or combinations thereof. Exemplary framework structures and coupling chemistries are described below in the context of the conjugate.

  In various embodiments, aptamers are covalently linked to each other or to a framework structure. In such embodiments, the aptamer may be directly linked (ie, without intervening chemical groups) and a spacer (eg, a cup that provides some degree of physical isolation between aptamers or between aptamers and the framework structure). It may be indirectly linked via a ring agent or a covalent bond chain). As discussed above in the context of the conjugate, aptamers are covalently linked to each other or to the framework by any number of chemical bonds, including but not limited to amide, ester, ether, isourea and imine bonds. It will be understood that this can be done.

In various embodiments, two or more aptamers are non-covalently bound to each other or to a framework structure. In some specific embodiments, the non-covalent dissociation constant (K _d ) in human serum is less than 1 pmol / L. For example, aptamers can be non-covalently bound to each other or to a framework by non-covalent ligand-receptor pairs well known in the art (eg, but not limited to biotin-avidin based pairs). In such embodiments, one member of the ligand receptor pair is covalently bound to one aptamer and the other member of the pair is covalently bound to the other aptamer or framework. When aptamers (or aptamers and frameworks) are combined, strong non-covalent interactions between the ligand and its receptor result in aptamers being non-covalently bound to each other (or to the framework). Typical ligand / receptor pairs include protein / cofactor and enzyme / substrate pairs. In addition to commonly used biotin / avidin pairs, such pairs include, but are not limited to, biotin / streptavidin, digoxigenin / anti-digoxigenin, FK506 / FK506 binding protein (FKBP), rapamycin / FKBP, cyclophilin / cyclosporine, and A glutathione / glutathione transferase pair may be mentioned. Other suitable ligand / receptor pairs such as, but not limited to, glutathione-S-transferase (GST), c-myc, FLAG®, and Kessler pp. 105-152, Advances in Mutagenesis "Kessler ed., Springer-Verlag, 1990;" Affinity Chromatography: Methods and Protocols (Methods in Molecular Biology) ", Pascal Baillon ed., Humana Press, 2000; and Hermanson et al.," Immobilized Affinity Ligand Techniques Monoclonal antibodies in pairs with epitope tags such as those described in Academic Press, 1992 will be recognized by those skilled in the art.

3. It will be appreciated that in general any of the multivalent crosslinkers described above can be chemically modified, for example, to reduce undesirable properties.

i. Non-specific modification In US2007-0110811 we described the benefit of pegylating this in order to reduce the in vivo mitogenic activity of the lectin. Thus, in some specific embodiments, the multivalent crosslinker may be modified by covalent bonds with one or more compounds. Without limitation, this may be an activated PEGylation (PEG) agent (eg, but not limited to N-hydroxysuccinimide activated PEG, succinimidyl ester of PEG propionic acid, succinimidyl ester of PEG butanoic acid, Reagents such as succinimidyl esters of PEGα-methylbutanoate), other water-soluble but non-PEG-containing polymers (such as poly (vinyl alcohol)), such as reagents that can be easily coupled to lysine by the use of carbodiimide reagents, perfluoro compounds It may be accompanied by a reaction with the above. Other suitable compounds will be readily recognized by those skilled in the art, for example, by referring to a comprehensive review that can be found in, for example, “Chemical Reagents for Protein Modification” by Lundblad, CRC Press, 3rd edition, 2004. Like.

  In general, the compound (s) can be attached to a polyvalent crosslinker (eg, mitogen lectin) by any of several conjugation methods known to those skilled in the art (eg, by amine, carboxyl, hydroxyl or sulfhydryl groups). ) Can be combined. The possible covalent bonds are similarly diverse (for example, amide bonds, carbamate bonds, ester bonds, thioether bonds, ether bonds, disulfide bonds, etc.). In some specific embodiments, an appropriate reactive group is grafted onto a multivalent cross-linking agent (eg, mitogen lectin) by introducing an appropriate amino acid by site-directed mutagenesis known in the art. obtain. For example, PEG is conveniently attached via an amino or carboxyl group. Examples of amino acid residues having a free amino group include lysine residues and the N-terminal amino acid residue. Examples of amino acid residues having a free carboxyl group include aspartic acid residues, glutamic acid residues, and the C-terminal amino acid residue. The sulfhydryl group found in cysteine residues can also be used as a reactive group to attach PEG (or other compounds). In a preferred embodiment, PEG is covalently linked to an amino group, particularly a free amino group found on a lysine residue.

Numerous methods for conjugating PEG directly to proteins are described by Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9: 249-304, 1992; Francis et al., Intern. J. et al. of Hematol. 68: 1-18, 1998; US Pat. No. 4,002,531; US Pat. No. 5,349,052; WO 95/06058; and WO 98/32466. One example of such a method uses tresylated monomethoxypoly (ethylene glycol) (MPEG), which is made by reacting MPEG with tresyl chloride (ClSO ₂ CH ₂ CF ₃ ). Tresylated MPEG reacts with exposed amine groups on the lectin. One skilled in the art will recognize that the present invention is not limited to any particular PEGylating agent (or compound) and can identify other suitable compounds well known in the art.

  In some specific embodiments, PEG (or other compound) can be attached to the multivalent crosslinker via an intervening linker. For example, US Pat. No. 5,612,460 discloses a urethane linker for linking PEG to a protein. PEG is MPEG-succinimidyl succinate, MPEG activated with 1,1′-carbonyldiimidazole, MPEG-2,4,5-trichlorophenyl (penyl) carbonate, MPEG- It can be coupled by reaction with compounds such as p-nitrophenol carbonate and various MPEG-succinate derivatives. Several additional PEG derivatives and reaction chemistries for conjugating PEGs to proteins are described in WO 98/32466 and other patents, such as Shearwater of Huntsville, AL; Nektar Therapeutics of San Carlos, CA; and / or Enzon Pharmaceuticals , NJ. A catalog describing a series of suitable PEG compounds and chemical reactions can be obtained from such commercial PEG suppliers (see, eg, Nektar Advanced PEGylation CATALOG 2004).

  In various embodiments, the N-terminal α-amine and / or ε-aminolysine group of the polypeptide-based cross-linking agent alters the distribution of charges and any tertiary and quaternary effects associated with such alterations. For this purpose, it may be succinylated and / or acetylated. For example, a polypeptide can be succinylated by reaction with excess succinic anhydride in saturated sodium acetate buffer. Acetylation can be performed using the same procedure except that acetic anhydride is used as the modifier. For example, if the protein is concanavalin A, both acetylation and succinylation not only increase the density of negative charges within the polypeptide, but also result in synthesis as a dimer rather than a tetramer at physiological pH. (See, eg, Agrawal et al., Biochemistry. 7: 4211-4218, 1968 and Gunther et al., Proc. Natl. Acad. Sci, (USA) 70: 1012-1016, 1973). Also, as a result, the in vivo safety profile of the resulting substance is greatly improved. Also, as a result, the in vivo safety profile of the resulting substance is greatly improved.

ii. Binding Site Modification In some specific embodiments, it may be advantageous to use a more specific and alternative method of modifying the multivalent crosslinker. In particular, the inventors have found that in certain low molecular weight conjugates of the present disclosure, when combined with highly pegylated lectins made using high molecular weight PEG reagents (> 5 kDa), an insoluble drug delivery system is It was found that it was not formed. This presents a challenge because we have previously found that low molecular weight PEG (<5 kDa) is much less effective in reducing the mitogenic activity of lectins. Without wishing to be limited to any particular theory, larger PEG groups can sterically hinder the binding and network formation with smaller low valent conjugates, while larger high valent conjugates are hindered. It will not be possible. In view of this, the inventors have devised an alternative non-PEG solution to improve the safety profile of lectin-based crosslinkers. The inventors have achieved this by specifically targeting and modifying the saccharide binding site of the lectin. For example, by reacting mannose ligand directly into the concanavalin A binding site and purifying unreacted material by high affinity ligand chromatography, we are comparable to that of the best pegylated lectin. A crosslinker with a safety profile could be synthesized. Without wishing to be limited to any particular theory, this functional concept is that the cell surface has a defined saccharide affinity, valency and ligand density, but the conjugate has all these properties tailored by design. It is thought that it can be done. Thus, incorporation of mannose within the lectin binding site completely abolishes the ability of the cross-linking agent to bind to cells, thereby aggregating or stimulating the cells, but with a higher density of high affinity ligands. Upon incorporation into the conjugate, gel formation is still possible. In some specific embodiments, the incorporation of individual PEG chains with a low degree of PEGylation and low MW has been newly designed to stabilize multivalent lectins in solution under various extreme storage conditions It can be used to obtain a manufacturable and safe functional cross-linking agent that complements the conjugate.

  In general, a binding site-modified lectin includes at least one covalently bound affinity ligand that can associate with one of the lectin binding sites. In various embodiments, the modified lectin may contain only one covalently linked affinity ligand. In various embodiments, the lectin can include one covalently bound affinity ligand per binding site. Typically, a multivalent lectin is one that contains two or four binding sites (eg, a dimer or tetramer of a monovalent lectin), although the disclosure also includes three, five, or Also included are lectins having more binding sites. The present disclosure also encompasses lectins having more than one covalently bound affinity ligand per binding site. Furthermore, the present disclosure encompasses a substance comprising a mixture of lectins containing different numbers of covalently bound affinity ligands and / or lectins containing unmodified lectins.

  Any affinity ligand can be used for this purpose, as long as it can associate with the binding site of the lectin once covalently bound to the lectin. Typically, an affinity ligand contains a recognition element that, once bound within the binding site, allows the lectin binding site and reactive linker (which allows the affinity ligand to be covalently bound to the lectin. To interact with).

Recognition Elements Any recognition element that can compete for binding to the lectin cognate ligand (eg, glucose or mannose in the case of Con A) can be used for the affinity ligands of the present disclosure. In various embodiments, the recognition element includes a carbohydrate. In some specific embodiments, the carbohydrate is a natural sugar (eg, glucose, fructose, galactose, mannose, arabinose, ribose, xylose, etc.). In some specific embodiments, the carbohydrate is a modified carbohydrate (eg, 2′-fluororibose, 2′-deoxyribose, hexose, etc.). In some specific embodiments, the recognition element is glucose, sucrose, maltose, mannose, derivatives thereof (eg, glucosamine, mannosamine, methyl glucose, methyl mannose, ethyl glucose, ethyl mannose, etc.) and / or their high The following combinations (eg, linear and / or branched bimannose, linear and / or branched trimannose, etc.).

  Other exemplary carbohydrates will be recognized by those skilled in the art. In particular, depending on the application, it is possible that any one of the above carbohydrates (eg any one of the formula IVa or IVb carbohydrates) can be used in the context of the conjugate's affinity ligand. It will be understood. In some specific embodiments, the recognition element comprises a monosaccharide. In some specific embodiments, the recognition element comprises a disaccharide. In some specific embodiments, the recognition element comprises a trisaccharide. In some embodiments, the recognition element is one that includes a carbohydrate and one or more amine groups. In some embodiments, the recognition element is aminoethyl glucose (AEG). In some embodiments, the recognition element is aminoethyl mannose (AEM). In some embodiments, the recognition element is aminoethylbimannose (AEBM). In some embodiments, the recognition element is aminoethyltrimannose (AETM). In some embodiments, the recognition element is β-aminoethyl-N-acetylglucosamine (AEGA). In some embodiments, the recognition element is aminoethyl fucose (AEF). In other embodiments, the recognition element is D-glucosamine (GA).

  In various embodiments, the recognition element is one that comprises a polysaccharide, glycopeptide or glycolipid. In some specific embodiments, the recognition element comprises 2-10 carbohydrate moieties, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 such moieties. The terminal and / or internal residues of a polysaccharide, glycopeptide or glycolipid can be selected based on the sugar specificity of the lectin of interest (eg, Goldstein et al., Biochem. Biophys. Acta 317: 500-504, 1973 and Lis). Et al., Ann. Rev. Biochem. 55: 35-67, 1986).

  In various embodiments, the recognition element for a particular lectin / exogenous target molecule combination can be selected based on experimentation. According to such embodiments, one or more recognition elements are screened based on the relative binding affinity for the lectin as compared to the exogenous target molecule. In some specific embodiments, a library of carbohydrates and / or polysaccharides is screened in this manner. A suitable recognition element is one that exhibits a detectable level of competition with the exogenous target molecule, but does not compete so strongly that it completely prevents binding between the lectin and the exogenous target molecule. In some specific embodiments, different recognition elements are tested by examining the effect of various affinity ligands on the properties of the relevant lectin (eg, the ability to deter aggregation and / or its substance setpoint (hereinafter , And discussed in more detail in each example). In some specific embodiments, the recognition element takes into account a conjugate that is combined with a modified lectin (eg, the conjugate displaces the recognition element from the binding site, thereby forming a cross-linked substance). Selected).

Reactive linker The affinity ligand can be covalently attached to the lectin in any manner. Most methods involve associating a ligand recognition element with a lectin binding site and then reacting a reactive linker with the lectin. In some specific embodiments, the reactive linker may be attached to the recognition element at a position that does not substantially interfere with the binding properties of the recognition element. For example, if the recognition element is a carbohydrate or polysaccharide, the linker can be attached to the C1, C2 or C6 position of the terminal sugar. In some specific embodiments, the linker can be attached to the C1 position. The C1 position, also referred to as the anomeric carbon, can be linked to the linker in an α or β conformation. In some specific embodiments, the linker is attached as an α anomer at the C1 position.

  In some specific embodiments, photoexcitable linkers can be used. For example, Beppu et al. Biochem. 78: 1013-1019, 1975 reported a method of activating an aryl azide linker using ultraviolet light to form a double bond between concanavalin A and a saccharide derivative in the binding site. Fraser et al., Proc. Using succinylated concanavalin A. Natl. Acad. Sci. (USA) 73: 790-794, 1976, similar results were recorded. In addition, lysine and Houston, J .; Biol. Chem. 258: 7208-7212, 1983, using a photoexcitable derivative of galactose and using a similar procedure. Also, photoexcitable derivatives of complex glycopeptide ligands with higher affinity for lectins than carbohydrates and disaccharides are described by Baenziger et al. Biol. Chem. 257: 4421-4425, 1982. Such derivatives were made by covalently attaching a photoexcitable group to the peptide portion of a glycopeptide ligand.

  In general, any photoexcitable linker such as aryl, purine, pyrimidine, or alkyl azide, diazo or diazirine group, benzophenone, or nitrobenzene can be used. A more comprehensive list of potentially useful photoexcitable linkers can be found in Fleming, Tetrahedron 51: 12479-12520, 1995 and Brunner, Annu. Rev. Biochem. 62: 483-514, 1993 and Wong, S .; S. “Chemistry of Protein Conjugation and Cross-Linking” (1993), CRC Press, New York, pp. Look at 168-194.

  In various embodiments, the photoexcitable linker can include a diazirine group. Photoexcitation of the diazirine group with ultraviolet (UV) light produces a reactive carbene intermediate, which is an addition reaction with any amino acid side chain or peptide backbone within the linker. Can form a double bond. Long wavelength UV light (about 320-370 nm, preferably about 345 nm) is typically used for activation of diazirine (see, eg, Suchanek et al., Nat. Methods 2: 261-268, 2005). . In various embodiments, the photoexcitable linker can include an aryl azide group.

  When the aryl azide group is exposed to UV light, a nitrene group is formed, which nitrene group is added to the double bond, inserted into the C—H and N—H sites, or subsequent ring expansion. And can react with primary amines as nucleophiles. The latter reaction path becomes dominant when primary amines are present in the sample. Without limitation, for substituted aryl azides (eg, having a hydroxy or nitro group) long wavelength UV light (about 320-370 nm, preferably about 366 nm) is believed to be very efficient, but unsubstituted For aryl azides, short wavelengths are considered very efficient. Suitable UV light sources are commercially available from, for example, Pierce, Rockford, IL.

For example, in various embodiments, the affinity ligand of the general formula _(IX): R e those in and give a -L ^1, wherein, _{R e} is recognition element, -L ¹ is a reactive linker . In some certain embodiments, R _e is carbohydrate moiety. In some specific embodiments, R _e is a glucose or mannose moiety covalently linked to the linker at the C1 position.

In some specific embodiments, -L ¹ is of the general formula (Xa):

(Where:
R ³ is independently selected from the group consisting of hydrogen, —OH, —NO ₂ , and halogen (eg, —F or —Cl);
X ^L is C _1-20 hydrocarbon chain optionally substituted saturated or unsaturated straight or branched chain of covalent bonds or where divalent, wherein one or more X ^L In some cases, the methylene units of -0-, -S-, -N (R ')-, -C (O)-, -C (O) O-, -OC (O)-,- N (R ′) C (O) —, —C (O) N (R ′) —, —S (O) —, —S (O) ₂ —, —N (R ′) S 0 ₂ —, —SO ₂ is substituted with N (R ′) —, a heterocyclic group, an aryl group, or a heteroaryl group;
Each R 'present is independently hydrogen, a suitable protecting group, or an acyl, arylalkyl, aliphatic, aryl, heteroaryl or heteroaliphatic moiety)
It may have.

In any case where a chemical variable is displayed attached to a bond that intersects a ring bond (eg, as indicated by R ³ above), this is because one or more such variable moieties are , Means optionally attached to the ring having crossed bonds. Each R ³ group on such a ring may be attached at any suitable position; this is generally understood to mean that the group is attached instead of a hydrogen atom on the parent ring. This includes the possibility that two R ³ groups may be bound to the same ring atom. Further, when more than one R ³ group is present on the ring, each may be the same or different from the other R ³ groups attached, each group having the same identifier Is defined independently of other groups that can be attached to another part of the same molecule.

In some specific embodiments, the —N ₃ group is in the meta position. In some specific embodiments, the —N ₃ group is in the ortho position. In some specific embodiments, the —N ₃ group is in the para position.

In certain embodiments, 1, 2, 3, 4 or 5 methylene units of X ^L is replaced independently optionally. In certain embodiments, ^{X L} _{is, C 1-10, C 1-8, C} 1-6, C 1-4, C 2-12, C 4-12, C 6-12, C 8 _-12 or _{C 10-12} are constructed with a hydrocarbon chain, wherein one or more methylene units of ^{X L} is independently optionally, -O -, - S -, - N (R ')-, -C (O)-, -C (O) O-, -OC (O)-, -N (R') C (O)-, -C (O) N (R ')-, Replaced with —S (O) —, —S (O) ₂ —, —N (R ′) S 0 ₂ —, —SO ₂ N (R ′) —, a heterocyclic group, an aryl group, or a heteroaryl group; ing. In some embodiments, one or more methylene units of X ^L is replaced by a heterocyclic group. In some embodiments, one or more methylene units of X ^L is replaced by the triazole moiety. In certain embodiments, one or more methylene units of X ^L is -C (O) - are replaced with. In certain embodiments, -C one or more methylene units of ^{X L (O) N (R} ') - has been replaced with. In certain embodiments, one or more methylene units of X ^L is replaced by -O-.

In some embodiments, ^XL is

It is.

In some embodiments, ^XL is

It is.

In some embodiments, ^XL is

It is.

In some embodiments, ^XL is

It is.

In some embodiments, ^XL is

It is.

In some embodiments, ^XL is

It is.

In some specific embodiments, -L ¹ is of the general formula (Xb):

(Wherein, X ^L is as defined above in Formula Xa,
R ⁴ is hydrogen, C ₁ -C ₆ alkyl or —CF ₃ )
Can be.

  In some specific embodiments, non-photoexcitable linkers can be used. For example, US Pat. Nos. 5,239,062 and 5,395,924 describe linkers that can be activated by changes in pH or temperature. Exemplary reactive linkers discussed include cyanuric acid (Kay et al., Nature 216: 514-515, 1967) or dichloro-S-triazine (2-amino-4,6-dichloro-S-triazine and the like (Kay Biochim. Biophys. Acta 198: 276-285, 1970)) and 2,4-dichloro-6-methoxy-S-triazine (Lang et al., J. Chem. Soc. Perkin 1: 2189-2194, 1977) and the like. And those that can be introduced into the affinity ligand using the above reagents. Reactive linkers with NHS-esters or aldehydes that can react primarily with terminal amines (such as those found in lysine) can also be used.

  In various embodiments, the reactive linker for a particular lectin / target molecule combination can be selected based on experimentation. According to such embodiments, several affinity ligands having the same recognition element and different linkers (eg, different length linkers, different reactive group linkers, different hydrophobicity linkers, etc.) Screening based on effects on properties (eg, based on ability to suppress aggregation and / or substance setpoints (discussed below and in more detail in this example)).

ii. Degree of Modification In general, the number of compounds (ie, the degree of substitution) bound to each multivalent crosslinker depends on the nature of the crosslinker, the nature of the compound (s), the number of available reactive sites and Varies depending on reaction conditions. For example, each concanavalin A subunit contains 12 lysine residues. As a result, when concanavalin A is PEGylated with a compound that reacts with a lysine residue, each subunit has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 subunits. To the compound. Conversely, each subunit of concanavalin A contains only one glucose binding site. Thus, when concanavalin A is reacted with a compound that reacts at the binding site, each subunit is covalently bound to only one such compound. A method for measuring the degree of substitution is described by Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9: 249-304, 1992.

  In preferred embodiments, chemical modification of the multivalent crosslinker can be optimized using multiple compounds and multiple reaction conditions (eg, differing reagent concentrations, pH, temperature, etc.). Preferred compounds and reaction conditions are such that undesirable properties (eg, mitogenic activity) are reduced compared to unmodified crosslinkers, while desired properties (eg, binding affinity) are not substantially impaired. . For example, using a robotic automated handling device, a series of modified compositions can be prepared using various compounds and various reaction conditions. Using routine orthogonal experimental techniques, one skilled in the art will be able to screen the properties of the treated composition. In some specific embodiments, an additional number of orthogonal optimizations are performed to further refine the preferred compounds and reaction conditions for the preferred conditions.

  In one embodiment, optimal reaction conditions are identified by separating the treated composition by electrophoresis, preferably by denaturing SDS-PAGE electrophoresis. In various embodiments, compositions comprising a uniformly modified crosslinker are preferred. Such preferred compositions have a weak band in the molecular weight of the unmodified crosslinker as measured by SDS-PAGE.

4). Purification of the crosslinker In various embodiments, the polyvalent crosslinker (whether chemically modified or not) may be further processed to improve its properties. Thus, in some specific embodiments, a composition comprising a multivalent crosslinker can be purified to remove protein fragments, unmodified components, and the like. In general, such separation can be performed based on physical properties (eg, charge; molecular weight; and / or size) and / or chemical properties (eg, binding affinity for a target molecule). In some specific embodiments, optimal removal may be performed by combining two or more methods that depend on such property differences. In one embodiment, such separation is performed under denaturing conditions. For example, unmodified or partially modified crosslinkers can be removed by ion exchange chromatography based on their net charge. Using gel filtration chromatography, differentially modified crosslinkers can be identified based on size. Affinity chromatography is another method that can be used to remove unmodified or partially modified crosslinkers. This approach takes advantage of the difference in binding affinity of modified, partially and unmodified crosslinkers for specific target molecules.

5. Crosslinking Agent Characterization In various embodiments, the multivalent crosslinking agent (whether chemically modified or not) may be screened or further tested to confirm or characterize its properties. . Representative assays include affinity assays, aggregation assays, T cell mitogen activity assays, T cell viability assays, antigenicity assays, and the like.

  Affinity assays involve passing a multivalent cross-linking agent through an affinity column (eg, a resin with a target molecule) and determining the elution conditions required to remove the cross-linking agent from the column. possible. Equilibrium dialysis can also be used as is known in the art. A setpoint assay may also be used in which the crosslinker is combined with one or more conjugates of the present disclosure and then contacted with various concentrations of the target molecule. Preferably, the binding affinity of the chemically modified crosslinker is at least 75% of that of the unmodified crosslinker. More preferably, the binding affinity is at least 85%, even more preferably at least 95% of that of the unmodified crosslinker.

  In some specific embodiments, the minimum aggregation concentration (MAC) of the multivalent crosslinker can be measured using an aggregation assay. For example, in some specific embodiments, MAC can be measured using rabbit erythrocytes, as described in US 20070110811. The inventors have found that in the case of chemically modified lectins, high MAC values are strongly correlated with low mitogenic activity. In some specific embodiments, the modified crosslinker may have a higher MAC than the unmodified crosslinker. Preferably, the MAC is 25 times that of the unmodified crosslinker. More preferably, the MAC is 50 times, even more preferably more than 100 times that of the unmodified crosslinker. In some specific embodiments, the modified crosslinker exhibits a MAC greater than 4 ug / ml in a 2% v / v suspension of formaldehyde stabilized rabbit erythrocytes. Preferably, the MAC is greater than 6 ug / ml, more preferably greater than 10 ug / ml, even more preferably greater than 25 ug / ml.

  Mitogen activity assays typically involve contacting a subject composition with a T cell culture (eg, PBMC cells) for a period of time and then measuring the level of T cell proliferation. Various methods are known for measuring cell proliferation. In one embodiment, the cell density can be measured spectrophotometrically at 450 nm. In another embodiment, indirect measurements can be obtained by detecting a reduction in MTT at 570 nm (see, eg, Ohno et al., J. Immunol. Methods 145: 199-203, 1991). In preferred embodiments, the level of cell proliferation is measured using a tritium labeled thymidine incorporation assay. One skilled in the art will recognize that other suitable methods may be used and that the present invention is not limited to any particular proliferation assay. In some specific embodiments, the T cell mitogenic activity of the modified crosslinker is less than 50% of the T cell mitogenic activity of the unmodified crosslinker. Reduction in T cell mitogenic activity can be assessed by performing a relative thymidine incorporation assay over a range of crosslinker concentrations (eg, 0.01, 0.1, 1, 10, 100 and 1000 ug / ml). . In a preferred embodiment, the thymidine incorporation assay is performed on a sample containing approximately 500,000 PBMCs. The mitogenic activity of the test composition (eg, modified composition) is then expressed as a percentage of the maximum unmodified mitogenic activity. The% of maximum unmodified mitogenic activity is obtained by dividing the maximum CPM (counts per minute) value of the test composition at all measured concentrations by the maximum CPM value of the unmodified composition at all measured concentrations. . Preferably, a test composition with reduced mitogenic activity induces T cell proliferation levels that are at least 50% lower than the unmodified composition. More preferably, the level is at least 75% lower, even more preferably at least 90%, 95% or 99% lower.

  T cell viability is determined by using similar experiments, adding trypan blue to a T cell culture and counting a representative sample of the cells (either taking up the trypan or still excluding the trypan. That is, i.e., those that are blue and those that are not). The percent viability is then calculated by dividing the number of cells that have excluded trypan (“non-blue” live cells) by the total number of cells counted (“blue” dead cells + “non-blue” live cells). To do. One skilled in the art will recognize that other suitable methods can be used and that the present invention is not limited to any particular viability assay. In some specific embodiments, the modified cross-linking agent exhibits a percent cell viability greater than 10% when assayed with PBMC at a concentration of 500,000 cells / ml at 100 ug / ml. Preferably, the percent cell viability is greater than 25%, more preferably greater than 50%, and even more preferably greater than 90%.

Cross-linked material body When the conjugate and cross-linking agent are combined in the absence of the exogenous target molecule, a non-covalent cross-linked material body is formed. In various embodiments, the material can be prepared by self-assembly by mixing a solution of a crosslinker and a conjugate in an aqueous solution. In various embodiments, the particles of the mass can be prepared by inverse emulsification. This can be done by adding the aqueous solution to a mixture of hydrophobic liquid and surfactant and stirring the mixture, as described in more detail in US 2004/0202719.

  Once formed, the cross-linked material can be used for a variety of applications. When the substance is placed in the presence of a free exogenous target molecule, the molecule competes for the interaction between the crosslinker and the conjugate. For free exogenous target molecules above a certain concentration, the level of competition is such that the substance begins to degrade as conjugates are released from the surface. In various embodiments, the extent and / or rate of release increases as the concentration of exogenous target molecule increases. As a result, the conjugate is released from the material in a manner that is directly related to the local concentration of the exogenous target molecule.

  In general, the release characteristics of the substance will depend on the nature and conditions of the crosslinker, conjugate, exogenous target molecule (eg, pH, temperature, nature and concentration of the endogenous molecule bound to the crosslinker, etc.). If the affinity of the cross-linking agent is much greater for the conjugate than for the exogenous target molecule, the substance releases only the conjugate at a high exogenous target molecule concentration. As the relative affinity of the cross-linking agent for the conjugate decreases, the release of the conjugate from the material occurs at a lower exogenous target molecule concentration. Also, the release characteristics of the substance can be adjusted by changing the relative amount of crosslinker relative to the conjugate. A higher ratio of crosslinker to conjugate results in a substance that releases the conjugate at higher exogenous target molecule concentrations. A lower ratio of crosslinker to conjugate results in a substance that releases the conjugate at a lower exogenous target molecule concentration. It will be appreciated that, depending on the application, such changes can produce substances that respond to a wide variety of exogenous target molecule concentrations.

In various embodiments, the cross-linked material is insoluble when placed in HEPES buffered saline at 37 C and pH 7 (25 mM HEPES containing 150 mM NaCl). In various embodiments, the cross-linked material remains substantially insoluble when exogenous target molecules are added to the buffer to a threshold concentration referred to as the set point. Above the set point, the cross-linked material shows an increase in the extent and rate of release of the conjugate. It will be appreciated that this transition can occur rapidly and can occur slowly over a series of concentrations near the set point. In general, the desired setpoints and transitions depend on the nature of the exogenous target molecule and the intended application of the substance. In particular, if the substance is designed to respond to increased levels of a particular exogenous target molecule, the desired setpoint is determined based on the PK profile of the exogenous target molecule (especially the C _max ). obtain. The amount of exogenous target molecule present in the patient depends on the route of administration, dose and schedule, and further on the delivery means (for exogenous target molecules delivered orally, for example, immediate release, It will be appreciated that sustained release and / or delayed release formulations may be used).

  It will be appreciated that the desired setpoint for any exogenous target molecule can be readily determined for a variety of different applications. Also, the setpoint can be based on a particular patient (eg, based on patient gender, patient having abnormally low or high levels of exogenous target molecule absorption, etc.) or application (eg, high frequency criteria It will also be appreciated that drug delivery systems designed to release at low levels may need to be adjusted for) that may require lower threshold concentrations than systems designed to release at low frequencies.

  It will be appreciated that a substance having a desired set point can be made by routine experimentation using the materials and methods described herein. For example, the same crosslinker and conjugate can be combined to make a series of materials with increasing ratio of crosslinker to conjugate (w / w). Such materials cover a range of setpoints. Once a lead material body with an appropriate setpoint is identified, the process may be repeated using a more precise resolution to obtain an optimized material body. Alternatively (or additionally), the same conjugate may be combined with a plurality of different cross-linking agents with increasing affinity for the conjugate. This yields multiple bodies with a range of setpoints, which may be further refined (eg, by changing the w / w ratio of the crosslinker to the conjugate). Alternatively, the process may be initiated by combining the same crosslinker with multiple different conjugates. In various embodiments, the conjugate can have various affinities for the cross-linking agent (eg, as a result of including different affinity ligands). In various embodiments, the conjugates can include the same affinity ligand but have different molecular weights (eg, as a result of different conjugate frameworks).

  In various embodiments, the material remains substantially insoluble when placed in normal human serum at 37 C for 6 hours using USP Dissolution Test Method II at 50 rpm. In various embodiments, when using USP Dissolution Test Method II at 50 rpm and placing in normal human serum at 37 C for 6 hours, less than 1%, less than 2%, less than 4%, less than 6% Less than 8% or less than 10%. In various embodiments, the substance of the present disclosure uses USP Dissolution Test Method II at 50 rpm and is 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, It can remain substantially insoluble when placed in HEPES buffered saline at pH 7 at 37 C with 350 or 400 mg / dL glucose. In various embodiments, USP Dissolution Test Method II is used at 50 rpm and is 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, or 400 mg / dL. When placed in 37 C pH 7 HEPES buffered saline with glucose for 6 hours, less than 1, 2, 4, 6, 8 or 10% substance dissolves.

Use In another aspect, the present disclosure provides a method of using the material. In general, the substance can be used to controllably release the conjugate in response to an exogenous target molecule.

  In various embodiments, the substance can be used to controllably deliver a drug to a patient. The present invention encompasses treatment of a disease or condition by administering a substance of the present disclosure. The substance may be used for the treatment of any patient (eg, dog, cat, cow, horse, sheep, pig, mouse, etc.), but is most preferably used for human treatment. The substance can be administered to the patient by any route. In general, the most suitable route of administration will depend on various factors such as the nature of the disease or condition being treated, the nature of the drug, the nature of the target molecule, the physical condition of the patient, and the like. In general, the present disclosure includes oral, intravenous, intramuscular, intraarterial, subcutaneous, intracerebroventricular, transdermal, rectal, intravaginal, intraperitoneal, transsurface (by powder, ointment or drops), oral administration Or administration as an oral or nasal spray or aerosol. General considerations in formulating and manufacturing pharmaceutical compositions for such various routes are described, for example, in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Co. , Easton, PA, 1995.

In various embodiments, the substance can be administered subcutaneously, for example, by injection. The material may be dissolved in a carrier to facilitate delivery. For example, the carrier can be an aqueous solution, including but not limited to, sterile water, saline, or buffered saline. In general, a therapeutically effective amount of a drug in conjugated form is administered. A “therapeutically effective amount” of a drug treats the disease or condition with a reasonable benefit / risk ratio (which involves a balance of drug efficacy and toxicity) (eg, the symptom is improved) A sufficient amount of drug is intended (such as delayed progression, suppressed recurrence, delayed onset, etc.). In general, therapeutic efficacy and toxicity can be determined by standard pharmacological procedures, in cell cultures, or using laboratory animals, for example, ED ₅₀ (dose that is therapeutically effective in 50% of treated subjects). And LD ₅₀ (dose lethal to 50% of treated subjects) can be determined. ED ₅₀ / LD ₅₀ represents the therapeutic index of the drug. In general, as is well known in the art, drugs with a large therapeutic index are preferred, but a small therapeutic index can be tolerated in the case of a serious disease or condition, especially if there are no alternative treatment options . The appropriate final selected dose range for administration to humans will be determined during the course of clinical trials.

  In various embodiments, the drug is insulin and the average daily dose of insulin ranges from 10 to 200 U, such as 25 to 100 U (wherein 1 unit of insulin is about 0.04 mg). In some specific embodiments, an amount of the substance with such an insulin dose is administered on a daily basis. In some specific embodiments, an amount of the substance with a dose of 5-10 times such an insulin dose is administered on a weekly basis. In some specific embodiments, an amount of the substance with a dose of 10 to 20 times such an insulin dose is administered on a biweekly basis. In some specific embodiments, an amount of the substance with a dose of 20 to 40 times such an insulin dose is administered monthly. One skilled in the art may apply this same approach to other approved drugs with known dose ranges (eg, any of the approved insulin sensitizers and insulin secretagogues described herein). It will be recognized that it is good.

  It will be appreciated that the total daily usage of the drug for any given patient will be determined by the attending physician within reasonable medical judgment. The specific therapeutically effective amount for any particular patient will vary according to various factors such as the disease or condition being treated; the activity of the specific drug used; the specific composition used; the age of the patient, Body weight, general health, sex and diet; administration period, route and excretion rate of specific drug used; duration of treatment; used in conjunction with or simultaneously with specific drug used Depending on the drug being used; as well as similar factors well known in the medical field. In various embodiments, the substance of the present disclosure can be administered more than once. For example, the present disclosure specifically provides for a continuous schedule (eg, once a day, once every two days, once a week, once every two weeks, once a month) by subcutaneous injection of the substance into a patient. Etc.).

  In some specific embodiments, the substance of the present disclosure can be used to treat hyperglycemia in a patient (eg, a mammalian patient). In some specific embodiments, the patient has diabetes. However, the method of the present invention is not limited to the treatment of diabetic patients. For example, in some specific embodiments, the substance can be used to treat hyperglycemia in patients with infections associated with impaired glycemic control. In some specific embodiments, the substance can be used to treat diabetes.

  In various embodiments, the substance of the present disclosure can be administered to a patient receiving at least one additional therapeutic agent. In various embodiments, the at least one additional therapy is intended to treat the same disease or disorder as the substance being administered. In various embodiments, the at least one additional therapy is intended for the treatment of side effects of the active ingredient. These two or more treatments can be administered in the same time frame, in overlapping time frames, or in non-overlapping time frames while the patient is benefiting from both therapies. May be. The two or more therapies may be administered on the same schedule or on different schedules while the patient is benefiting from both therapies. The two or more therapies may be administered in the same formulation or different formulations throughout the patient's benefit from both therapeutic agents. In some specific embodiments, a single substance of the present disclosure may contain more than one drug for treating the same disease or disorder. In some specific embodiments, two or more separate substances of the present disclosure may be administered (as a mixture or separately) containing different drugs for treating the same disease or disorder. In some specific embodiments, a non-conjugated secondary drug may be included in the substance body of the present disclosure (ie, simply mixed with a component of the substance and shared with the cross-linked substance body). Drug not bound). For example, in some particular embodiments, more than one type of antidiabetic agent can be administered to a subject using any such approach. Some specific exemplary embodiments of this inventive approach are described in more detail below in the context of insulin-related treatments, but the foregoing suggests that the benefits of such combined approaches of other therapies are You will be recognized.

  Insulin sensitivity-improving drugs (eg, biguanides such as metformin, glitazone) act by increasing the patient's response to a given amount of insulin. Accordingly, the dose of the insulin-based substance of the present disclosure required for a patient receiving an insulin sensitivity improving drug is less than that of the same patient not receiving the improving drug. Thus, in some specific embodiments, the substance comprising an insulin conjugate can be administered to a patient who is also receiving treatment with an insulin sensitizer. In various embodiments, the substance of the present disclosure can be administered at up to 75% of the usual dose required in the absence of an insulin sensitizer. In various embodiments, up to 50, 40, 30 or 20% of the normal dose can be administered.

  Insulin resistance is a disorder in which normal insulin levels are insufficient to produce a normal insulin response. For example, insulin resistant patients may require high doses of insulin to overcome the resistance and provide a sufficient glucose lowering effect. In such cases, insulin doses that can induce hypoglycemia usually in patients with low resistance do not have an equivalent glucose lowering effect in highly resistant patients. Similarly, the substance of the present disclosure is only effective for this subclass of patients if a high level of insulin-conjugate is released in the appropriate time frame. In some specific embodiments, treatment of this subclass of patients can be facilitated by combining the two approaches. Accordingly, in some specific embodiments, conventional insulin-based therapeutic agents are used to obtain a baseline level of insulin, and when the patient is in need, the substance of the invention is administered to provide control of insulin supplementation. Thus, in some specific embodiments, a substance comprising an insulin conjugate can be administered to a patient who is also undergoing treatment with insulin. In various embodiments, insulin can be administered at up to 75% of the normal dose required in the absence of the substance of the present disclosure. In various embodiments, up to 50, 40, 30 or 20% of the normal dose can be administered. This combination approach also applies to insulin resistant patients who are taking insulin secretagogues (eg, sulfonylureas, GLP-1, exendin-4, etc.) and / or insulin sensitizers (eg, biguanides, such as metformin, glitazone). It will be appreciated that it can be used.

Once administered, the substance can be stimulated by administration of a suitable exogenous target molecule. In some specific embodiments, a stimulating amount of an exogenous target molecule is administered. As used herein, a “stimulating amount” of an exogenous target molecule is an amount sufficient to cause the release of some amount of conjugate from a previously administered substance. It will be appreciated that any of the aforementioned administration methods for the substance apply equally to the exogenous target molecule. It will also be appreciated that the method of administration of the substance and the exogenous target molecule may be the same or different. In various embodiments, the method of administration is different (eg, for illustrative purposes, the substance can be administered weekly by subcutaneous injection, but the exogenous target molecule is administered orally daily). . Oral administration of exogenous target molecules is particularly valuable because it helps patient compliance. It will be appreciated that in general the release profile of the conjugate from the substance is related to the PK profile of the exogenous target molecule. Thus, the release profile of the conjugate can be adapted by controlling the PK profile of the exogenous target molecule. As is well known in the art, the PK profile of the exogenous target molecule can be adapted based on the dose, route, frequency and formulation used. For example, if a short and strong release of the conjugate is desired, an oral immediate release formulation can be used. In contrast, in cases where less intense release of the conjugate over the long term is desired, an oral sustained release formulation can be used instead. General considerations in the formulation and manufacture of immediate and sustained release formulations are described, for example, in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Co. , Easton, PA, 1995. In general, it will be appreciated that the set point of the substance is less than the C _maximum of the exogenous target molecule formulation where conjugate release occurs. For example, in various embodiments, the setpoint may be less than C _max 10, 20, 30, 40, ₅₀ , 60, 70, 80 or 90%.

  It will also be appreciated that the relative administration frequency of the substance of the present disclosure and the exogenous target molecule may be the same or different. In some specific embodiments, the exogenous target molecule is administered more frequently than the substance. For example, in some specific embodiments, the substance can be administered daily, while the exogenous target molecule is administered more than once a day. In some specific embodiments, the substance may be administered twice a week, weekly, biweekly or monthly, whereas the exogenous target molecule is administered daily. In some specific embodiments, the substance is administered monthly and the exogenous target molecule is administered twice weekly, weekly, or biweekly.

Kit In another aspect, the present disclosure provides a kit comprising a cross-linking agent, a conjugate, and other reagents for preparing a substance. For example, a kit can include separate containers that contain multiple conjugates and multiple crosslinkers. When the kit conjugate and the cross-linking agent are mixed, a cross-linked material is formed. In various embodiments, the substance is designed for subcutaneous delivery and the kit includes a syringe. In various embodiments, the kit can include a syringe pre-filled with a cross-linked material. In addition, the kit may include instructions for mixing the conjugate and a crosslinking agent to obtain a crosslinked substance. The kit may also contain an exogenous target molecule formulation, for example, an oral dosage form such as a capsule or tablet.

  In yet another aspect, the present disclosure provides a library of conjugates and / or crosslinkers. Such a library can be particularly useful for the production of substances having a desired set point. In various embodiments, the library can include multiple crosslinkers that result in different setpoints in the same conjugate. In various embodiments, the library can further comprise one or more conjugates that form a cross-linked material body with a cross-linking agent within the library. Where the library contains more than one such conjugate, different conjugates can have different molecular weights, different numbers of affinity ligands per conjugate molecule and / or different affinity ligands. In various embodiments, the library can include one or more conjugates that include more than one type of affinity ligand. In various embodiments, the library can include multiple conjugates that result in different setpoints with the same crosslinker. In various embodiments, the library can further include one or more cross-linking agents that form a cross-linked material body with the conjugate within the library.

Examples Example 1-Synthesis of α-Methyl-Mannose Stimulated Substances Exemplary conjugates were prepared according to the method of Example 8 using TSB-C4 as the backbone, AEBM as the affinity ligand, and NH ₂ -B1- as the drug. Synthesized with BOC2 (Al, B29) -insulin (Figures 1-2 for the structure of affinity ligands and conjugates, and Examples 3-7 for the methods used to prepare these starting materials. See 0.50 ml of 2.3 mg / ml conjugate solution (in 25 mM HEPES buffer (S14 buffer) containing 0.150 M sodium chloride at pH 8.2) was added to the centrifuge tube followed by 0.500 ml of 18 mg / ml. Immediate mixing with ml natural Con A (NCA) solution (in 25 mM HEPES buffer (S24 buffer) containing 0.150 M sodium chloride at pH 7.4) formed a dispersion of insoluble particles. The dispersion was left at room temperature for 20 minutes and then separated from the supernatant by centrifugation. The resulting cake was washed 5 times with 1.0 ml of 25 mM HEPES buffer (S24 buffer) containing 0.150 M sodium chloride at pH 7.4. After the last wash, residual insolubles were incubated overnight at 37C. The next day, residual particles were again isolated by centrifugation and washed once more in 1.0 ml S24. The insoluble material obtained was dispersed in a total volume of 0.30 ml (using S24) and reserved for later testing. This process may be scaled up directly to produce any amount of the desired product.

Example 2 α-Methyl-Mannose Stimulation in Nondiabetic Rats 0.300 ml of the formulation prepared in Example 1 was transformed into 3 normal male Sprague Dawley (SD) rats (Charles River Laboratories, Wilmington, Mass.) (Weight 400 Each of ~ 500g) was injected subcutaneously. Prior to injection of the formulation, blood glucose levels were measured using a Precision Xtra blood glucose meter (Abbott Laboratories, Alameda, Calif.) By collecting blood from the tail vein, and approximately 100 ul of serum was collected by collecting blood from the tail vein. Obtained and assayed for background levels of insulin. During the test period, food was removed from the rat cage. Serum and blood glucose values were obtained at 30, 60, 90 and 120 minutes after injection. 120 minutes after the injection, an intraperitoneal injection of 25% w / v α-methyl-mannose solution was injected at a dose of 2 g / kg, and then the serum and blood glucose levels were 135 minutes and 150 minutes. , 180 minutes, 210 minutes, 240 minutes, and 300 minutes. Subsequently, serum insulin concentrations were measured using a standard curve generated with a pure insulin conjugate solution using a commercial ELISA kit (Human Insulin ELISA, Mercodia, Uppsala, Sweden). In this assay, endogenous rat insulin does not cross-react. Thus, all results obtained were only with exogenously administered insulin conjugate and not with endogenous rat insulin.

  FIG. 3 shows an approximately 4-fold increase in serum insulin concentration from baseline after an intraperitoneal α-methyl-mannose challenge test (IP (α-MM) TT), indicating α-methyl-mannose responsiveness in vivo. Indicates delivery. Furthermore, very few conjugates were released at physiologically normal blood glucose levels during the first 2 hours of the experiment, and virtually no hypoglycemia was induced prior to the introduction of α-methyl-mannose. It was. However, a significant hypoglycemic effect was exerted when exogenously delivered α-methyl-mannose induced a 4-fold increase in serum insulin conjugate concentration.

Example 3 Synthesis of TSB-C4 Framework Structure A solution of 1,3,5-benzenetricarbonyl chloride (1 g, 3.8 mmol) in dichloromethane (DCM) (5 mL) was added to 1N of ω-amino acid (3.1 eq). To a vigorously stirred solution of NaOH (25 mL) is added dropwise in an ice bath. The ice bath is removed and stirring is continued for 4 hours at room temperature. 2N HCl (about 15 mL) is added dropwise until approximately pH 2 and the resulting slurry is stirred for an additional 2 hours. The precipitate is filtered, washed with cold water (2 × 20 mL), air dried under vacuum and then dried overnight in a 60 C oven. The resulting white solid is used without further purification. Yield of each ω-amino acid (4-aminobutyric acid: yield 1.6 g, 91%; 6-aminocaproic acid: yield 1.9 g, 92%).

The above material was placed in DMSO (5 mL) containing N-hydroxysuccinimide (3.1 mmol, 3.1 eq) and N- (3-dimethylaminopropyl) -N-ethylcarbodiimide (EDCI, 3.6 mmol, 3.6 equivalents) is added at room temperature. 'The resulting solution is stirred for 24 hours, diluted with water (125 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic phases are washed with water (2 × 50 mL), brine (1 × 50 mL) and dried over MgSO ₄ . Evaporate the solvent and triturate the semi-solid residue with acetonitrile (10 mL). The solid is filtered, washed with cold solvent, air dried under vacuum and then dried overnight in a 60 C oven. The product does not contain urea by-products. Benzene-1,3,5-tricarboxy- (N-6-aminocaproic acid-NHS ester) amide (TSB-C6): 304 mg, 36%, mp 140-142C. Benzene-1,3,5-tricarboxy- (N-4-butyric acid-NHS-ester) amide (TSB-C4): 245 mg, 45%, mp 182-184C.

Example 4 Synthesis of Azidoethyl Mannose (AzEM) a. Synthesis of Bromoethyl Mannose DOWEX 50Wx4 resin (Alfa Aesar, Ward Hill, Mass.) Is decolorized by washing with deionized water. A mixture of 225 g D-mannose (1.25 mol; 1 eq, Alfa Aesar) and 140 g DOWEX 50Wx4 was added to 2.2 L 2-bromoethanol (30.5 mol, 25 eq; 124.97 g / mol; 1.762 g). BP = 150C; Alfa Aesar) and the stirred mixture is heated to 80C for 4 hours. The reaction is monitored by TLC (20% methanol / dichloromethane (DCM)). The reaction is complete after about 4 hours and then allowed to cool to room temperature. The solution is filtered to remove the resin and the resin is washed with ethyl acetate and DCM. Stripping the resulting filtrate in a rotary evaporator results in an amber oil.

  The amber oil is purified on silica gel (4 kg silica-filled DCM) as follows. The crude is dissolved in DCM, loaded onto the column and then eluted with 2 × 4 L 10% methanol / DCM; 2 × 4 L 15% methanol / DCM; and 3 × 4 L 20% methanol / DCM. . Product containing fractions (based on TLC) are pooled and stripped to dryness to give 152 g of 1-α-bromoethyl-glucose (42%).

b. Conversion of Bromoethyl Mannose to Azidoethyl Mannose (AzEM) A 5 L round bottom three-necked flask equipped with a heating mantle, overhead stirrer and thermometer is charged with 150 g of bromoethyl mannose (525 mmol). This oil was dissolved in 2 L water and treated with 68.3 g sodium azide (1.05 mol, 2 eq; 65 g / mol; Alfa-Aesar) followed by 7.9 g sodium iodide (52.5 mmol). 0.08 eq .; 149.89 g / mol; Alfa-Aesar) and warm the solution to 50 C and stir overnight. The solution is cooled to room temperature and concentrated to dryness on a rotary evaporator. The solid residue is digested with 3 × 500 mL of 5: 1 (volume ratio) CHCl ₃ : MeOH at 40C. The combined organic portions are filtered and evaporated to dryness to give didoethylmannose as an off-white color.

c. Repurification of azidoethylmannose 32 g of azidoethylglucose is placed in 100 mL of water. The turbid solution is filtered through a glass microfiber filter (Whatman GF / B). The filtrate becomes a solid when evaporated on a rotary evaporator. This solid is taken up in methanol (100 mL) and the turbid solution is filtered again through a glass microfiber filter. The resulting pale yellow filtrate is stripped under vacuum to a solid.

  Put this solid in a minimum of methanol (50 mL) and slowly add ethyl acetate (150 mL) under stirring. Allow the heavy slurry to cool and filter. The solid is air dried (hygroscopic) and placed in a 60 C oven overnight. When the mother liquor is evaporated under vacuum, it becomes a yellow gum.

Example 5 Synthesis of Azidoethyl Mannobiose (AzEBM) The AzEM compound of Example 4 was selectively protected with benzene dimethyl ether and purified by column chromatography followed by reaction with benzyl bromide. -Α- (2-Azidoethyl) -4,6-benzaldehyde diacetal-3-benzyl-mannopyranoside is obtained. This product was then glycosylated with 1-α-bromo-2,3,4,6-tetrabenzoylmannopyranoside using a triflate silver chemistry under strict anhydrous conditions to provide a protected form. An azidoethyl mannobiose product is obtained. The intermediate product is then deprotected to remove the benzoyl group and yield AzEBM.

Example 6 Synthesis of Azidoethyl Mannobiose (AEBM) The azide-terminated compound of Example 5 is easily hydrogenated at room temperature by using a palladium / carbon catalyst, a small amount of acetic acid, and ethanol as the solvent, The corresponding amine-terminated compound is obtained. The process is identical to that described below for AETM, but it will be understood by those skilled in the art that the amounts of reagents, solvents, etc. should be increased or decreased depending on the number of moles of saccharide-ligand to be hydrogenated. Like.

a. Man (α-1,3) -Mann (α-1.6) -α-1-aminoethylmannopyranoside (“Aminoethyltrimannose”, AETM)
To a solution of 5.3 g (9.25 mmol) man (α-1,3) -man (α-1.6) -α-1-azidoethylmannopyranoside in 100 mL water / 50 mL ethanol was added 0. .8 g of 5% Pd / C was added. This vigorously stirred suspension was hydrogenated at 30-40 psi for 48 hours or until no starting material was seen by TLC (SG, methanol, SM R _f 0.75, Pdt R _f 0.0, PMA vis .). The suspension was filtered through celite, which was rinsed with ethanol (2 × 50 mL) and the filtrate was concentrated in vacuo.

HPLC of this material (C18, ₃ % acetonitrile / 97% 0.1% H ₃ PO ₄ , 220 nm, 2 ml / min) gave uv adsorption of the injected column void material, Rt 2.5 min, indicating benzoate ester. It was done.

  The filtrate was diluted with 70 mL water and 12 mL 1N NaOH and the solution was stirred overnight at room temperature (HPLC: column void Rt no uv material at 2.5 min, uv material at Rt 10.5 min co-administered with benzoic acid. Elution). 2g activated carbon for decolorization was added and the stirred suspension was heated to 80C, cooled to room temperature and filtered through celite. The pH of the filtrate was adjusted to 8.0 with 2N HCl and the colorless solution was concentrated in vacuo to about 50% volume.

This solution was loaded onto a resin column (Dowex 50W, 50 g) and washed with water until the elution fraction was at neutral pH (6 × 75 mL) to remove residual acid byproduct (if any). The amine product was washed from the column with 0.25N ammonium hydroxide (6 × 75 mL) and the fractions containing amine product-ninhydrin detection were combined and concentrated in vacuo to 25-30 mL. This concentrated solution was added dropwise to 300 mL of stirred ethanol and stirring was continued for another 2 hours. The product was filtered, washed with fresh ethanol (2 × 50 mL) and air dried until constant weight. The resulting white amorphous solid was further dried in a vacuum oven at 80 C for 5 hours to obtain 4.1 g of a white granular solid (TY 5.1 g). In NMR, there were no aromatic protons. ¹ H NMR 300 MHz (D ₂ O) δ 5.08 (s, 1H), 4.87 (s, 1H), 4.81 (s, 1H), 4.8-3.6 (m, 18H) 2.9 (m, 2H).

Example _{7-NH 2 -B1-BOC2 (} A1, B29) - dissolved in the synthesis typical synthesis of insulin, powdered insulin 4g (Sigma Aldrich, St.Louis, MO ) was added to a solution of anhydrous DMSO of 100ml And 4 ml of triethylamine (TEA) is added. The solution is stirred for 30 minutes at room temperature. Next, 1.79 ml (2.6 eq) of di-tert-butyl-dicarbonate / THF solution (Sigma Aldrich, St. Louis, MO) is slowly added to the insulin-TEA solution and mixed for approximately 1 hour. The reaction is quenched by adding 4 ml stock solution containing 250 ul ethanolamine in 5 ml DMSO and mixing for 5 minutes. After quenching, the entire solution is poured into 1600 ml of acetone and mixed gently with a spatula. Next, an 8 × 400 μl aliquot of a 18.9% HCl: water solution is dropped onto the surface of the mixture to precipitate the reacted insulin. The precipitate is then centrifuged and the supernatant decanted into a second beaker while the precipitate cake is secured. To the supernatant, a further 8 × 400 μl aliquot of 18.9% HCl: water solution is dropped onto the surface of the mixture to obtain a second precipitate of reacted insulin. This second precipitate is centrifuged and the supernatant is discarded. The combined centrifugal cake of the two precipitation steps is washed once with acetone and then vacuum dried at room temperature to obtain a crude powder. This typically includes 60% of the desired BOC2 product and 40% of the BOC3 material.

  Pure B0C2-insulin is isolated from the crude powder using a preparative reverse phase HPLC method. Buffer A is deionized water containing 0.1% TFA, and Buffer B is acetonitrile containing 0.1% TFA. The crude powder is dissolved at 25 mg / ml in a 70% A / 30% B mixture, syringe filtered and then injected into the column. Prior to purification, the column (Waters SymmetryPrep C18, 7 um, 19 × 150 mm) is equilibrated with a 70% A / 30% B mobile phase using a Waters DeltaPrep 600 system at 15 ml / min. Approximately 5 ml of the crude powder solution was injected onto the column over 5 minutes at a flow rate of 15 ml / min and then linear from 70% A / 30% B to 62% A / 38% B in the next 3.5 minutes. Use a gradient and hold in this state for an additional 2.5 minutes. Using this method, the desired BOC2 peak elutes at approximately 10.6 minutes, followed immediately by the BOC3 peak. Once collected, the solution is rotary concentrated to remove acetonitrile and lyophilized to obtain pure BOC2-insulin powder. Specifics are confirmed by LC-MS (HT Laboratories, San Diego, CA) and conjugation sites are examined by N-terminal sequencing (Western Analytical, St. Louis, MO).

Example 8-Conjugate Synthesis After the TSB-C4 framework structure is dissolved at 60 mM in 1.0 ml anhydrous DMSO, 400 ul (excess) triethylamine (TEA) is added. The solution is stirred at room temperature for 10 minutes at high speed. Next, NH ₂ -B1-BOC2 (A1, B29) -insulin (MW = 6,008 g / mol) is separately dissolved in 7.9 ml of DMSO at a concentration of 7.4 mM. Add dropwise to the framework / DMSO / TEA solution over 10 minutes, then mix for 2 hours at room temperature, and then react the remaining activated ester with the amine functionalized AEBM affinity ligand as follows. A 370 mM solution of the sex ligand is prepared in an appropriate volume of dry DMSO, and once dissolved, several reactions are added, equal to 3 times the number N-1 of initially activated ester groups. For example, if there are N = 3 initially activated ester groups per framework structure, (3 × (3-1) × 60 mM / 370 mM) = 0.993 m If there are N = 4 initially activated ester groups per framework structure, (3 × (4-1) × 60 mM / 370 mM) = 1.46 ml affinity Add the ligand solution, etc. After adding the affinity ligand solution, the solution is stirred for an additional hour at room temperature to ensure the end of the reaction.

The resulting solution is then superdiluted 10-fold in 20 mM (pH 5.0) HEPES buffered saline solution containing 0.150 M NaCl, and then adjusted to a final pH of 8.0 with dilute HCl. This aqueous solution is first purified by size exclusion (using an appropriate solid phase for the desired separation of conjugated and unconjugated materials). This solution passed through the column void volume is then concentrated to approximately 10 ml using an appropriately sized ultrafiltration membrane. This solution is further purified to give the desired product (using preparative reverse phase HPLC on a Waters C8, 7 um, 19 × 150 mm column). Buffer A is deionized water containing 0.1% TFA, and Buffer B is acetonitrile containing 0.1% TFA. Prior to purification, the column is equilibrated with a Waters DeltaPrep 600 system at 80 ml A / 20% B mobile phase at 15 ml / min. Approximately 5 ml of the crude solution was injected onto the column at a flow rate of 15 ml / min over 2 minutes, followed by a linear gradient from 80% A / 20% B to 75% A / 25% B for the next 5 minutes. Then, for the next 22 minutes, use a gentle linear gradient from 75% A / 25% B to 62% A / 38% B. The desired peak retention time depends on the drug used, the framework structure and the affinity ligand. Once collected, the solution is spin concentrated to remove acetonitrile and lyophilized to give pure conjugate, the specifics of which can be confirmed by LC-MS (HT Laboratories, San Diego, Calif.). _NH starting material 2 -B1-BOC2 (A1, B29 ) - Insulin, since only one Phe-B1-terminal no free amine groups, Phe-B1, each deprotected final product by N-terminal sequencing Is the only insulin conjugation site for the framework structure.

Example 9-Conjugate of Formula (I) In this example, Formula (I):

Several exemplary conjugates of are described.

  Still other embodiments of this conjugate, as well as intermediates and methods of making the conjugate, are described in US Provisional Application No. 61 / 162,105 (filed March 20, 2009) and the corresponding PCT application (January 2010). 27th application). The entire contents of these related applications are incorporated herein by reference.

  In some particular embodiments, the conjugate of formula (I) may include one or more of the following exemplary groups.

R ^x
In some specific embodiments, R ^x is hydrogen. In some specific embodiments, R ^x is optionally substituted C _1-6 alkyl. In some specific embodiments, R ^x is optionally substituted C _1-3 alkyl. In some specific embodiments, R ^x is optionally substituted methyl. In some specific embodiments, R ^x is —CH ₃ .

Z ¹
In some specific embodiments, Z ¹ is an optionally substituted divalent C _1-10 , C _1-8 , C _1-6 , C _1-4, or C _1-2 hydrocarbon. Is a chain. In some specific embodiments, Z ¹ is — (CH ₂ ) —, — (CH ₂ CH ₂ ) —, — (CH ₂ CH ₂ CH ₂ ) —, — (CH ₂ CH ₂ CH ₂ CH _{2). ) -, - (CH 2 CH} 2 CH 2 CH 2 CH 2) -, or _{- (CH 2 CH 2 CH 2} CH 2 CH 2 CH 2) - a. In some specific embodiments, Z ¹ is — (CH ₂ ) — or — (CH ₂ CH ₂ ) —. In some specific embodiments, Z ¹ is — (CH ₂ ) —. In some specific embodiments, Z ¹ is — (CH ₂ CH ₂ ) —. In some specific embodiments, Z ¹ is — (CH ₂ CH ₂ CH ₂ ) —. In some specific embodiments, Z ¹ is — (CH ₂ CH ₂ CH ₂ CH ₂ ) —.

In some specific embodiments, Z ¹ is an optionally substituted divalent C _1-10 hydrocarbon chain, wherein ¹ , 2 or 3 methylene units of Z ¹ are optional Independently selected, —S—, —O—, —NR ^a —, — (C═NR ^a ) —, — (C═O) —, — (S═O) —, —S (═O) One or more groups selected from _2- , — (CR ^b ═CR ^b ) —, — (N═N) —, an optionally substituted arylene moiety or an optionally substituted heteroarylene moiety. Has been replaced. In some specific embodiments, Z ¹ is an optionally substituted divalent C _1-10 hydrocarbon chain, wherein ¹ , 2 or 3 methylene units of Z ¹ are optionally Independently substituted with one or more groups selected from —S—, —O—, —NR ^a —, — (C═NR ^a ) —, or — (C═O) —. In some specific embodiments, Z ¹ is —CH ₂ CH ₂ NH (C═O) C (CH ₃ ) ₂ —, —CH ₂ CH ₂ N (C═NH) (CH ₂ ) ₃ S— _{^{, -CH (R f) 2,}} -CH 2 CH (R f) 2, -CH 2 CH 2 CH (R f) 2 -, - CH 2 S-, or _-CH 2 _CH 2 is S-, wherein Wherein R ^f is an optionally substituted aliphatic, an optionally substituted heteroaliphatic, an optionally substituted aryl, an optionally substituted heteroaryl (eg, In some specific embodiments, R ^f is an optionally substituted aryl; in some specific embodiments, R ^f is phenyl). In some specific embodiments, Z ¹ is —CH ₂ CH ₂ NH (C═O) C (CH ₃ ) ₂ — or —CH ₂ CH ₂ N (C═NH) (CH ₂ ) ₃ S— It is. In some specific embodiments, Z ¹ is —CH ₂ CH ₂ NH (C═O) C (CH ₃ ) ₂ —. In some specific embodiments, Z ¹ is —CH ₂ CH ₂ N (C═NH) (CH ₂ ) ₃ S—.

Y ¹
In some specific embodiments, Y ¹ is a fragment of a free radical initiator. Such fragments include halogen, —OR ^e , —SR ^e , optionally substituted aliphatic, optionally substituted heteroaliphatic, optionally substituted aryl, and optionally substituted. Are included in the definition of Y ¹ .

In some specific embodiments, Y ¹ is hydrogen, halogen, or an initiator fragment. In some specific embodiments, Y ¹ is hydrogen or halogen. In some specific embodiments, Y ¹ is hydrogen or bromine.

X ¹
In some specific embodiments, X ¹ is —OR ^c . In some specific embodiments, X ¹ is a mixture of —OR ^c and —N (R ^d ) ₂ . In some specific embodiments, X ¹ is —N (R ^d ) ₂ .

W ¹ and

In some specific embodiments,

Is a covalent single bond.

In some specific embodiments, W ¹ is covalently attached to the polymer via an amino group. In some specific embodiments, W ¹ is covalently attached to the polymer via a primary amino group.

For example, in some specific embodiments, the group

Is the group

In which the [drug-NH-] or [drug-N =] group is a drug directly conjugated by a primary amino group. In other embodiments, the drug may include a pendant amino group-terminated spacer group (eg, an alkylene group, an arylene group, a heteroarylene group, an ester bond, an amide bond, etc.). The latter embodiment makes it possible to increase the distance between the active part of the drug and the polymer.

r
In some specific embodiments, r is an integer from 10 to 25 (inclusive). In some specific embodiments, r is an integer from 15 to 25 (inclusive). In some specific embodiments, r is an integer from 20 to 25 (inclusive). In some specific embodiments, r is an integer from 5 to 20 (inclusive). In some specific embodiments, r is an integer from 10 to 20 (inclusive). In some specific embodiments, r is an integer from 15 to 20 (inclusive). In some specific embodiments, r is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25. In some specific embodiments, r is 5. In some specific embodiments, r is 10. In some specific embodiments, r is 15. In some specific embodiments, r is 20. In some specific embodiments, r is 25.

In some specific embodiments, the group:

The group:

(Wherein the sum of (g + t) is equal to r). In some specific embodiments, g and t in each case are independently integers from 1 to 24 (inclusive), but the sum of (g + t) is 5 or more and 25 or less. . In some specific embodiments, g and t are about 1:10, 1: 9, 1: 8, 1: 7, 1: 6, 1: 5, 1: 4, 1: 3, 1: 2. Or in a ratio of 1: 1 (g: t). In some specific embodiments, g and t are about 1:10, 1: 9, 1: 8, 1: 7, 1: 6, 1: 5, 1: 4, 1: 3, or 1: It exists in the ratio of 2 (g: t).

Exemplary Conjugates In some specific embodiments, Formula (I-a1):

Can be used.

In certain embodiments, formula (I-a2):

Can be used.

In certain embodiments, formula (I-b1):

Can be used.

In certain embodiments, formula (I-b2):

Can be used.

In some specific embodiments, the formula (I-cl):

Can be used.

In certain embodiments, formula (I-c2):

Can be used.

In any of these exemplary conjugates, the group:

The group:

(Wherein the sum of (g + t) is equal to r, respectively). In some specific embodiments, r is 10. In some specific embodiments, r is 20.

Conjugate Characterization The conjugate can be analyzed by any analytical method such as nuclear magnetic resonance (eg, ¹ H NMR); molecular weight and polydispersity as gel permeation chromatography (GPC); and acid groups per chain. Can be characterized by Fourier transform infrared spectroscopy (FTIR) or acid titration.

  In some specific embodiments, the framework of the conjugate (ie, free of affinity ligands or drugs) has a molecular weight of less than 10,000 Da, such as in the range of about 100 to about 10,000 Da. In some specific embodiments, the conjugate framework has a molecular weight in the range of about 300 to about 5,000 Da. In some specific embodiments, the conjugate framework has a molecular weight in the range of about 500 to about 2,500 Da. In some specific embodiments, the conjugate framework has a molecular weight in the range of about 1,000 to 2,000 Da. In some specific embodiments, the conjugate framework has a molecular weight in the range of about 200-1,000 Da. In some specific embodiments, the conjugate framework has a molecular weight in the range of about 300-800 Da.

  In some specific embodiments, a mixture of conjugates is made. The conjugates may have the same molecular weight or different molecular weights in this mixture. In one embodiment, the polydispersity of the mixture is less than 1.5. In one embodiment, the polydispersity of the mixture is less than 1.25.

Example 10-Conjugate of Formula (II) In this example, Formula (II):

Several exemplary conjugates of are described.

  Other embodiments of this conjugate, as well as intermediates and methods of making this conjugate, are described in US Provisional Application No. 61 / 147,878 (filed Jan. 28, 2009), US Provisional Application No. 61/159. , 643 (filed on March 12, 2009), US Provisional Application No. 61 / 162,107 (filed on March 20, 2009), US Provisional Patent Application No. 61 / 163,084 (March 2009) No. 61 / 219,897 (filed Jun. 24, 2009), U.S. Patent Provisional Application No. 61 / 223,572 (filed Jul. 7, 2009), U.S. Patent Provisional See Application No. 61 / 252,857 (filed Oct. 19, 2009) and the corresponding PCT application (filed Jan. 27, 2010). The entire contents of these related applications are incorporated herein by reference.

In some embodiments, the present disclosure provides general formula (IIa-1):

A conjugate of

For example, in some embodiments, the present disclosure provides the formula:

A conjugate of

In some embodiments, the present disclosure provides general formula (IIa-2):

A conjugate of

For example, in some embodiments, the present disclosure provides the formula:

A conjugate of

In some embodiments, the present disclosure provides general formula (IIa-3):

A conjugate of

For example, in some embodiments, the present disclosure provides the formula:

A conjugate of

Conjugate Characterization The conjugate can be analyzed by any analytical method, for example, nuclear magnetic resonance (eg, ¹ H NMR); molecular weight and polydispersity as gel permeation chromatography (GPC); Fourier transform infrared spectroscopy (FTIR) ) And the like.

  In some specific embodiments, the framework of the conjugate (ie, does not include affinity ligands, drugs or detectable labels) has a molecular weight of less than 10,000 Da, such as in the range of about 100 to about 10,000 Da. Have In some specific embodiments, the conjugate framework has a molecular weight in the range of about 300 to about 5,000 Da. In some specific embodiments, the conjugate framework has a molecular weight in the range of about 500 to about 2,500 Da. In some specific embodiments, the conjugate framework has a molecular weight in the range of about 1,000 to 2,000 Da. In some specific embodiments, the conjugate framework has a molecular weight in the range of about 200-1,000 Da. In some specific embodiments, the conjugate framework has a molecular weight in the range of about 300-800 Da.

Example 11 Synthesis of Azidophenyl-Sugar Modified Con A This example and the following examples describe the preparation of several exemplary binding site modified lectins that can be used to prepare the materials of the present disclosure. To do.

  Unless otherwise noted, all steps were performed at room temperature. First, 5.0 g of natural Con A (Sigma-Aldrich, St. Louis, MO), 150 mM sodium chloride, 2 mM calcium chloride, 2 mM manganese chloride and 0.1% w / v sodium azide (S28 buffer). It was dissolved in 200 ml of 10 mM (pH 5.0) acetate buffer solution, and insoluble matter (if any) was separated by centrifugation and / or filtration. We have found that various natural Con A commercial preparations contain recognizable concentrations of inhibitory saccharide, which in some specific embodiments is removed prior to photoaffinity modification. Is done. For this purpose, the solution was purified twice through a Biogel-P6 size exclusion column (using S28 mobile phase). Finally, the resulting solution was diluted with S28 to a final volume of 1L. Under mild stirring conditions, 0.4 g hydroquinone (Sigma-Aldrich, St. Louis, MO) was added followed by 165 mg azidophenyl glucose (APG, PolyOrg Inc., Leominster, Mass.) Or azidophenylmannose (APM, PolyOrg Inc., Leominster, MA) was added. The solution was stirred at 4C in the dark for 1 hour at the lowest possible stirring rate. After 1 hour of stirring, additional insoluble material (if any) was removed by centrifugation and / or filtration. 200 ml of this solution was poured into a 9 inch × 13 inch aluminum pan and reacted at 360 nm for 15 minutes at 4 C (also UV reaction) inside a CL-1000 UV cross-linking furnace (UVP, Upland, CA). May be performed using 302 nm light). After the reaction, further insoluble material (if any) was removed by centrifugation and / or filtration. The clarified solution was then purified once through a Biogel-P6 size exclusion column (Econopak, Bio-Rad Labs, Hercules, Calif.) (Using S28 mobile phase). The UV cross-linking reaction and the P6 purification process were then repeated until all the solutions had reacted. Finally, the combined P6 purified solution was concentrated to approximately 180 ml using a Pall tangential flow filtration cartridge device (Millipore, Billerica, Mass.) Equipped with an Omega 30K membrane. The resulting solution was clarified by centrifugation and / or filtration, passed through a 0.22 um filter, and subjected to affinity column purification.

Example 12-General Synthesis of Diazirine Photoreactive Ligand 0.9 mmol of aminoethyl (AE) functionalized saccharide ligand (eg, AEG, AEM, AEBM, AETM) is dissolved in 4 ml of anhydrous DMSO, then 1 When 6 ml of anhydrous triethylamine (TEA) was added, an emulsion was formed. In a separate container, 200 mg (0.9 mmol) NHS-diazirine (Thermo Fisher Scientific Inc., Rockford, IL) powder was dissolved in 4 ml anhydrous DMSO under dark conditions. Once dissolved, the NHS-diazirine solution was added dropwise to the AE-saccharide solution and then allowed to react overnight in the dark at room temperature. By overnight TLC analysis of the solution (50% ethanol: 50% ethyl acetate), co-elution of the UV signal (254 nm) of the diazirine moiety and the saccharide signal (sulfuric acid-ethanol staining), as well as the AE-functionalized saccharide from the base of the TLC Since the disappearance of the ligand (sulfuric acid-ethanol staining) was shown, the completion of the reaction was confirmed. The solution is then diluted in 80 ml of 25 mM HEPES solution (pH 5.0) containing 0.15 M sodium chloride, the pH is adjusted to pH 5 if necessary, and then the photoaffinity reaction with Con A Freeze until needed.

Example 13 Synthesis and Characterization of Sugar Functionalized Diazirine Con A All steps were performed at room temperature unless otherwise noted. First, 5.0 g of natural Con A (Sigma-Aldrich, St. Louis, MO), 150 mM sodium chloride, 2 mM calcium chloride, 2 mM manganese chloride and 0.1% w / v sodium azide (S28 buffer). It was dissolved in 200 ml of 10 mM (pH 5.0) acetate buffer solution, and insoluble matter (if any) was separated by centrifugation and / or filtration. We have found that various natural Con A commercial preparations contain recognizable concentrations of inhibitory saccharide, which in some specific embodiments is removed prior to photoaffinity modification. Is done. For this purpose, the solution was purified twice through a Biogel-P6 size exclusion column (using S28 mobile phase). Finally, the resulting solution was diluted with S28 to a final volume of 1L. Next, the volume of this solution was reduced from 1 L to 1/3 of the ligand volume using 1 × S28, and injected into a 1 L media bottle containing a stirring bar. Under mild stirring conditions, 0.4 g of hydroquinone (Sigma-Aldrich, St. Louis, MO) was dissolved in the dark. Next, 33 ml of the diazirine-saccharide conjugate obtained in Example 43 was added in 7 portions in the dark under mild stirring conditions. Once dissolved, the entire solution was incubated for an additional 10 minutes at 4C in the dark with gentle agitation. After 10 minutes of stirring, additional insoluble material (if any) was removed by centrifugation and / or filtration. 250 ml of this solution was poured into a 9 inch × 13 inch aluminum pan and allowed to react at 360 nm for 15 minutes at 4 C inside a CL-1000 UV crosslinking furnace (UVP, Upland, CA). After the reaction, further insoluble material (if any) was removed by centrifugation and / or filtration. The clarified solution was then purified once through a Biogel-P6 size exclusion column (Econopak, Bio-Rad Labs, Hercules, Calif.) (Using S28 mobile phase). The UV cross-linking reaction and the P6 purification process were then repeated until all the solutions had reacted. Finally, the combined P6 purified solution was concentrated to approximately 180 ml using a Pall tangential flow filtration cartridge device (Millipore, Billerica, Mass.) Equipped with an Omega 30K membrane. The resulting solution was clarified by centrifugation and / or filtration, passed through a 0.22 um filter, and subjected to affinity column purification.

Other Embodiments Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification or practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims (61)

A cross-linked substance comprising a multivalent cross-linking agent that binds to an exogenous target molecule, and a conjugate comprising a drug and two or more individual affinity ligands bound to a conjugate framework structure,
The two or more affinity ligands compete with the exogenous target molecule for binding to the cross-linking agent, and as a result of non-covalent interactions between the affinity ligand on different conjugates and the cross-linking agent. The conjugate is crosslinked in the body,
Cross-linked material body.   The substance body according to claim 1, wherein the exogenous target molecule contains a carbohydrate.   The substance body according to claim 2, wherein the exogenous target molecule is α-methyl-mannose.   The substance body according to claim 2, wherein the affinity ligand of the conjugate contains a carbohydrate.   5. The substance body according to claim 4, wherein the affinity ligand of the conjugate comprises a carbohydrate selected from glucose, mannose, glucosamine, mannosamine, methyl glucose, methyl mannose, ethyl glucose, and ethyl mannose.   5. The substance body according to claim 4, wherein the affinity ligand of the conjugate comprises bimannose or trimannose.   5. A substance according to claim 4, wherein the affinity ligand of the conjugate comprises aminoethyl glucose (AEG), aminoethyl mannose (AEM), aminoethyl bimannose (AEBM) or aminoethyl trimannose (AETM).   5. The substance according to claim 4, wherein the affinity ligand of the conjugate includes a saccharide and a linker, and the saccharide is covalently bonded to the linker via an anomeric carbon.   The substance body according to claim 8, wherein the anomeric carbon is an α anomer.   The substance body according to claim 1, wherein the polyvalent crosslinking agent comprises a polypeptide.   The substance body according to claim 10, wherein the polyvalent crosslinking agent contains a lectin.   The substance body according to claim 11, wherein the lectin is Con A lectin.   The substance body according to claim 11, wherein the lectin is chemically modified.   14. The substance body according to claim 13, wherein the lectin is PEGylated.   A lectin is covalently bound to a recognition element that competes with an exogenous target molecule and the affinity ligand of the conjugate for binding to the lectin, and the lectin binds to the recognition element. 14. The substance according to claim 13, wherein the substance has a higher affinity for the affinity ligand of the conjugate.   16. The substance according to claim 15, wherein the exogenous target molecule is a carbohydrate, and the affinity ligand and the recognition element of the conjugate both include a carbohydrate. A recognition element in the lectin has the formula:

(Where
R ³ is independently selected from the group consisting of hydrogen, —OH, —NO ₂ , and halogen;
X ^L is a covalent bond or a divalent straight or branched, optionally substituted C _1-20 hydrocarbon chain, where one of X ^L The above methylene units are optionally and independently -O-, -S-, -N (R ')-, -C (O)-, -C (O) O-, -OC (O)-. , -N (R ') C ( O) -, - C (O) N (R') -, - S (O) -, - S (O) 2 -, - N (R ') SO 2 -, Substituted with —SO ₂ N (R ′) —, a heterocyclic group, an aryl group, or a heteroaryl group;
Each R 'present is independently hydrogen, a suitable protecting group, or an acyl, arylalkyl, aliphatic, aryl, heteroaryl or heteroaliphatic moiety)
The substance body according to claim 15, which is covalently bonded using a photoexcitable linker. A recognition element in the lectin has the formula:

(Where
R ⁴ is hydrogen, C ₁ -C ₆ alkyl or —CF ₃ ;
X ^L is a covalent bond or a divalent linear or branched saturated or unsaturated, optionally substituted C _1-20 hydrocarbon chain, wherein one of X ^L In some cases, the above methylene units are independently -0-, -S-, -N (R ')-, -C (O)-, -C (O) O-, -OC (O)-, -N (R ') C (O ) -, - C (O) N (R') -, - S (O) -, - S (O) 2 -, - N (R ') S0 2 -, - Substituted with SO ₂ N (R ′) —, a heterocyclic group, an aryl group, or a heteroaryl group;
Each R 'present is independently hydrogen, a suitable protecting group, or an acyl, arylalkyl, aliphatic, aryl, heteroaryl or heteroaliphatic moiety)
The substance body according to claim 15, which is covalently bonded using a photoexcitable linker.   The substance body according to claim 10, wherein the polyvalent crosslinking agent comprises a peptide aptamer.   The substance body according to claim 1, wherein the polyvalent crosslinking agent comprises a polynucleotide aptamer.   The substance body according to claim 1, wherein the drug is an antidiabetic drug.   The substance body according to claim 1, wherein the drug is an insulin molecule.   The substance body according to claim 1, wherein the drug is an insulin sensitivity-improving drug.   The substance body according to claim 1, wherein the drug is an insulin secretagogue.   The substance body according to claim 1, wherein the conjugate framework structure is a polymer system.   The substance body according to claim 1, wherein the conjugate framework structure is not a polymer system.   The substance body according to claim 1, wherein the conjugate framework structure is branched or hyperbranched.   The substance body according to claim 1, wherein the conjugate framework structure comprises a polysaccharide. Conjugates have the general formula:

(Where
R ^x is hydrogen or optionally substituted C _1-6 alkyl;
Z ¹ is an optionally substituted divalent C _1-10 hydrocarbon chain, wherein ¹ , 2, 3, 4, or 5 methylene units of Z ¹ are optionally independently ^{Te, -S -, - O -,} - NR a -, - (C = NR a) -, - (C = O) -, - (S = O) -, - S (= O) 2 -, - (CR ^b = CR ^b )-,-(N = N)-, substituted with one or more groups selected from an optionally substituted arylene moiety or an optionally substituted heteroarylene moiety Wherein R ^a is hydrogen, optionally substituted aliphatic, optionally substituted heteroaliphatic, optionally substituted aryl, optionally substituted heteroaryl. or a suitable amino protecting group, R ^b is an aliphatic hydrogen, it is optionally substituted Heteroaliphatic is optionally substituted, heteroaryl which is substituted aryl or optionally substituted with optionally;
Each X ¹ present is independently —OR ^C or —N (R ^d ) ₂ , wherein R ^c is hydrogen, optionally substituted aliphatic, optionally substituted. A heteroaliphatic, optionally substituted aryl, optionally substituted heteroaryl, a suitable hydroxyl protecting group, cationic group, or affinity ligand, wherein each R ^d is independently hydrogen An optionally substituted aliphatic, an optionally substituted heteroaliphatic, an optionally substituted aryl, an optionally substituted heteroaryl, a suitable amino protecting group, or affinity A ligand, but at least two of the X ¹ present shall comprise an affinity ligand;
Y ¹ is hydrogen, halogen, optionally substituted aliphatic, optionally substituted heteroaliphatic, optionally substituted aryl, optionally substituted heteroaryl, —OR ^e or —SR ^e , wherein R ^e is hydrogen, optionally substituted aliphatic, optionally substituted heteroaliphatic, optionally substituted aryl, or optionally A heteroaryl substituted with
r is an integer from 5 to 25 (inclusive);
W ¹ is a drug;

Is equivalent to a covalent single bond or double bond)
The substance body according to claim 1, wherein Conjugates have the general formula:

(Where [(AT)]
Each represents a possible branch in the conjugate;
(AT) each represents a possible repeat in the branch of the conjugate;
-A- are each independently substituted with a covalent bond, carbon atom, heteroatom, or optionally selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, heteroaryl and heterocyclic. Is a group;
Each T present is independently a C _1-30 hydrocarbon chain that is covalently bonded or optionally substituted with a divalent linear or branched saturated or unsaturated, Wherein one or more methylene units of T are optionally independently, -O-, -S-, -N (R)-, -C (O)-, -C (O) O-,- OC (O) -, - N (R) C (O) -, - C (O) N (R) -, - S (O) -, - S (O) 2 -, - N (R) SO 2 _{-, - SO 2 N (R} ) -, heterocyclic group, have been replaced by an aryl group or a heteroaryl group;
Each R present is independently hydrogen, a suitable protecting group, or an acyl, arylalkyl, aliphatic, aryl, heteroaryl or heteroaliphatic moiety;
-B is an ^-T-L B -X;
Each X present is independently an affinity ligand;
Each L ^B present is independently a group derived from the covalent conjugation with either a covalent bond, or T and X;
-D is -T-L ^D -W;
Each W present is independently a drug;
Each existing L ^D, independently, is a group derived from a covalent conjugation to a is or T and W covalent bond;
k is an integer from 2 to 11 (inclusive) and specifies that there are at least two k-branches in the conjugate;
q is an integer from 1 to 4 (inclusive);
k + q is an integer from 3 to 12 (inclusive);
Each p present is independently an integer from 1 to 5 (inclusive);
Each n present is independently an integer from 0 to 5 (inclusive);
Each m present is independently an integer from 1 to 5 (inclusive);
Each v present is independently an integer from 0 to 5 (inclusive), but at each k-branch, at least one n present is ≧ 1, and at least one v present is ≧ 1)
The substance body according to claim 1, wherein   31. The substance according to claim 29 or 30, wherein the conjugate in which X and W are not present respectively has a molecular weight of less than 10,000 Da.   32. The substance of claim 31, wherein the conjugate, in which X and W are each absent, has a molecular weight in the range of about 300 to about 5,000 Da.   32. The substance of claim 31, wherein the conjugate, in which X and W are not present respectively, has a molecular weight in the range of about 300 to about 800 Da.   2. A substance according to claim 1 which is insoluble when placed in 25 mM HEPES buffer at 37 C, pH 7, comprising 150 mM NaCl and exogenous target molecules.   2. The substance of claim 1, wherein the conjugate is released from the substance at a rate dependent on the concentration of the exogenous target molecule or to a degree dependent on the concentration of the exogenous target molecule.   36. The substance of claim 35, wherein the exogenous target molecule is α-methyl-mannose.   37. A substance according to claim 36 which remains substantially insoluble when placed in normal human serum at 37C for 6 hours using USP dissolution test method II at 50 rpm.   37. The substance of claim 36, wherein less than 10% of the substance dissolves when placed in normal human serum at 37C for 6 hours using USP Dissolution Test Method II at 50 rpm.   37. Using USP dissolution test method II at 50 rpm and remaining substantially insoluble when placed in pH 7,25 mM HEPES buffer at 37 C containing 150 mM NaCl and 400 mg / dL glucose for 6 hours. The substance body described in 1.   <10% of the substance dissolves when placed in pH 7,25 mM HEPES buffer for 6 hours at 37 C containing 150 mM NaCl and 400 mg / dL glucose using USP Dissolution Test Method II at 50 rpm. 36. The substance body according to 36.   41. A method comprising administering to a patient a substance according to any one of claims 1 to 40 and subsequently administering a stimulating amount of an exogenous target molecule to the patient.   42. The method of claim 41, wherein the substance is administered by subcutaneous injection.   42. The method of claim 41, wherein the conjugate comprises an insulin molecule attached to the framework structure.   42. The method of claim 41, wherein the substance is administered such that the average daily dose of insulin molecules ranges from 10 to 200U.   45. The method of claim 44, wherein the substance is administered daily.   45. The method of claim 44, wherein the substance is administered weekly.   45. The method of claim 44, wherein the substance is administered monthly.   45. The method of claim 44, wherein the patient is also receiving an insulin sensitizer.   45. The method of claim 44, wherein the patient is also taking an insulin secretagogue.   42. The method of claim 41, wherein the substance and exogenous target molecule are administered by different routes.   51. The method of claim 50, wherein the substance is administered by subcutaneous injection and the exogenous target molecule is administered orally.   42. The method of claim 41, wherein the relative frequency of administration of the substance and the exogenous target molecule is different.   53. The method of claim 52, wherein the exogenous target molecule is administered more frequently than the substance.   54. The method of claim 53, wherein the substance is administered daily and the exogenous target molecule is administered more than once a day.   54. The method of claim 53, wherein the substance is administered twice a week, weekly, biweekly or monthly and the exogenous target molecule is administered daily.   54. The method of claim 53, wherein the substance is administered monthly and the exogenous target molecule is administered twice weekly, weekly, or biweekly.   54. The method of claim 53, wherein the substance is administered weekly and the exogenous target molecule is administered daily.   54. The method of claim 53, wherein the substance is administered monthly and the exogenous target molecule is administered daily.   54. The method of claim 53, wherein the substance is administered monthly and the exogenous target molecule is administered weekly.   60. The method according to any one of claims 41 to 59, wherein the exogenous target molecule is a carbohydrate.   61. The method of claim 60, wherein the exogenous target molecule is [alpha] -methyl-mannose.

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